Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates ∼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.

Specific intestinal microbiota has been shown to induce Foxp3+ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103+ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103+ DCs from Il10 −/−, Tlr2 −/−, and Myd88 −/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103+ DCs failed to induce IL-10 production from co-cultured Il27ra −/− T cells. B. breve treatment of Tlr2 −/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103+ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4+ T cells from wild-type mice, but not Il10 −/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.

Toxoplasma virulence factor ROP18 targets endoplasmic reticulum–bound transcription factor ATF6β in the host cell, leading to the detrimental loss of ATF6β through proteasome-dependent degradation.

IFN-γ mediates cellular innate immunity against an intracellular parasite, Toxoplasma gondii, by inducing immunity-related GTPases such as p47 IFN-γ–regulated GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), which also participate in antibacterial responses via autophagy. An essential autophagy protein, Atg5, was previously shown to play a critical role in anti–T. gondii cell-autonomous immunity. However, the involvement of other autophagy proteins remains unknown. In this study, we show that essential autophagy proteins differentially participate in anti–T. gondii cellular immunity by recruiting IFN-γ–inducible GTPases. IFN-γ–induced suppression of T. gondii proliferation and recruitment of an IRG Irgb6 and GBPs are profoundly impaired in Atg7- or Atg16L1-deficient cells. In contrast, cells lacking other essential autophagy proteins, Atg9a and Atg14, are capable of mediating the anti–T. gondii response and recruiting Irgb6 and GBPs to the parasites. Although IFN-γ also stimulates anti–T. gondii cellular immunity in humans, whether this response requires GBPs and human autophagy proteins remains to be seen. To analyze the role of human ATG16L1 and GBPs in IFN-γ–mediated anti–T. gondii responses, human cells lacking ATG16L1 or GBPs were generated by the Cas9/CRISPR genome-editing technique. Although both ATG16L1 and GBPs are dispensable for IFN-γ–induced inhibition of T. gondii proliferation in the human cells, human ATG16L1 is also required for the recruitment of GBPs. Taken together, human ATG16L1 and mouse autophagy components Atg7 and Atg16L1, but not Atg9a and Atg14, participate in the IFN-γ–induced recruitment of the immunity-related GTPases to the intracellular pathogen.

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.

Also known as Sqstm1, p62 is a selective autophagy adaptor with a ubiquitin-binding domain. However, the role of p62 in the host defense against Toxoplasma gondii infection is unclear. Here, we show that interferon γ (IFN-γ) stimulates ubiquitin and p62 recruitment to T. gondii parasitophorous vacuoles (PVs). Some essential autophagy-related proteins, but not all, are required for this recruitment. Regardless of normal IFN-γ-induced T. gondii clearance activity and ubiquitination, p62 deficiency in antigen-presenting cells (APCs) and mice diminishes the robust IFN-γ-primed activation of CD8(+) T cells that recognize the T. gondii-derived antigen secreted into PVs. Because the expression of Atg3 and Irgm1/m3 in APCs is essential for PV disruption, ubiquitin and p62 recruitment, and vacuolar-antigen-specific CD8(+) T cell activation, IFN-γ-mediated ubiquitination and the subsequent recruitment of p62 to T. gondii are specifically required for the acquired immune response after PV disruption by IFN-γ-inducible GTPases.

Ma et al. show that the Toxoplasma gondii polymorphic dense granule protein GRA6 triggers the activation of the host transcription factor NFAT4, thus affecting the host immune response and maximizing parasite virulence.

No abstract