Regulatory T Cells Promote Myositis and Muscle Damage in Toxoplasma gondii Infection.

The coordination of macrophage polarization is essential for the robust regenerative potential of skeletal muscle. Repair begins with a phase mediated by inflammatory monocytes (IM) and proinflammatory macrophages (M1), followed by polarization to a proregenerative macrophage (M2) phenotype. Recently, regulatory T cells (Tregs) were described as necessary for this M1 to M2 transition. We report that chronic infection with the protozoan parasite Toxoplasma gondii causes a nonresolving Th1 myositis with prolonged tissue damage associated with persistent M1 accumulation. Surprisingly, Treg ablation during chronic infection rescues macrophage homeostasis and skeletal muscle fiber regeneration, showing that Tregs can directly contribute to muscle damage. This study provides evidence that the tissue environment established by the parasite could lead to a paradoxical pathogenic role for Tregs. As such, these findings should be considered when tailoring therapies directed at Tregs in inflammatory settings.

Tbet Expression by Regulatory T Cells Is Needed to Protect against Th1-Mediated Immunopathology during Toxoplasma Infection in Mice.

Toxoplasma gondii infection has proven to be an ideal model to understand the delicate balance between protective immunity and immune-mediated pathology during infection. Lethal infection causes a collapse of T regulatory cells (Tregs) mediated by the loss of IL-2 and conversion of Tregs to IFN-γ-producing cells. Importantly, these Tregs highly express the Th1 transcription factor Tbet. To determine the role of Tbet in Tregs, we infected Tbx21f/f -Foxp3YFPCre and control Foxp3YFPCre mice with the type II strain of T. gondii, ME49. The majority of Tbx21f/f -Foxp3YFPCre mice succumbed to a nonlethal dose. Notably, parasite burden was reduced in Tbx21f/f -Foxp3YFPCre compared with Foxp3YFPCre control mice. We found that Tbx21f/f -Foxp3YFPCre mice have significantly higher serum levels of proinflammatory cytokines IFN-γ and TNF-α, suggestive of a heightened immune response. To test if CD4+ T cells were driving immunopathology, we treated Tbx21f/f -Foxp3YFPCre mice with anti-CD4-depleting Abs and partially rescued these mice. Broad-spectrum antibiotic treatment also improved survival, demonstrating a role for commensal flora in immunopathology in Tbx21f/f -Foxp3YFPCre mice. RNA sequencing analysis reinforced that Tbet regulates several key cellular pathways, including leukocyte activation, regulation of lymphocyte activation, and cell cycle progression, that help to maintain fitness in Tregs during Th1 responses. Taken together, our data show an important role for Tbet in Tregs in preventing lethal immunopathology during T. gondii infection, further highlighting the protective role of Treg plasticity in controlling immune responses to infection and the microbiota.

The Toxoplasma Polymorphic Effector GRA15 Mediates Seizure Induction by Modulating Interleukin-1 Signaling in the Brain.

Toxoplasmic encephalitis can develop in individuals infected with the protozoan parasite Toxoplasma gondii and is typified by parasite replication and inflammation within the brain. Patients often present with seizures, but the parasite genes and host pathways involved in seizure development and/or propagation are unknown. We previously reported that seizure induction in Toxoplasma-infected mice is parasite strain dependent. Using quantitative trait locus mapping, we identify four loci in the Toxoplasma genome that potentially correlate with seizure development. In one locus, we identify the polymorphic virulence factor, GRA15, as a Toxoplasma gene associated with onset of seizures. GRA15 was previously shown to regulate host NF-κB-dependent gene expression during acute infections, and we demonstrate a similar role for GRA15 in brains of toxoplasmic encephalitic mice. GRA15 is important for increased expression of interleukin 1 beta (IL-1ß) and other IL-1 pathway host genes, which is significant since IL-1 signaling is involved in onset of seizures. Inhibiting IL-1 receptor signaling reduced seizure severity in Toxoplasma-infected mice. These data reveal one mechanism by which seizures are induced during toxoplasmic encephalitis. IMPORTANCE Inflammation in the brain caused by infections lead to seizures and other neurological symptoms. But the microbial products that induce seizures as well as the host pathways downstream of these factors are largely unknown. Using a nonbiased genetic screening approach, we identify 4 loci in the Toxoplasma genome that correlate with the induction of seizures in Toxoplasma-infected mice. One of these loci contains the gene, GRA15, which we demonstrate is associated with seizure development in toxoplasmic encephalitic mice. GRA15 accomplishes this in part by activating host pathways that lead to increased IL-1 receptor signaling and that inhibition of this signaling inhibits Toxoplasma-induced seizures.

Thermotolerance in the pathogen Cryptococcus neoformans is linked to antigen masking via mRNA decay-dependent reprogramming.

A common feature shared by systemic fungal pathogens of environmental origin, such as Cryptococcus neoformans, is their ability to adapt to mammalian core body temperature. In C. neoformans, this adaptation is accompanied by Ccr4-mediated decay of ribosomal protein mRNAs. Here we use the related, but thermo-intolerant species Cryptococcus amylolentus to demonstrate that this response contributes to host-temperature adaptation and pathogenicity of cryptococci. In a C. neoformans ccr4Δ mutant, stabilized ribosomal protein mRNAs are retained in the translating pool, and stress-induced transcriptomic changes are reduced in comparison with the wild type strain, likely due to ineffective translation of transcription factors. In addition, the mutant displays increased exposure of cell wall glucans, and recognition by Dectin-1 results in increased phagocytosis by lung macrophages, linking mRNA decay to adaptation and immune evasion.

Chronic infection stunts macrophage heterogeneity and disrupts immune-mediated myogenesis.

The robust regenerative potential of skeletal muscle is imperative for the maintenance of tissue function across a host of potential insults including exercise, infection, and trauma. The highly coordinated action of multiple immune populations, especially macrophages, plays an indispensable role in guiding this reparative program. However, it remains unclear how skeletal muscle repair proceeds in a chronically inflamed setting, such as infection, where an active immune response is already engaged. To address this question, we used a cardiotoxin injury model to challenge the reparative potential of chronically infected muscle. Compared with regenerating naive skeletal muscle, infected skeletal muscle exhibited multiple indicators of delayed muscle repair including a divergent morphologic response to injury and dysregulated expression of myogenic regulatory factors. Further, using both flow cytometric and single-cell RNA sequencing approaches, we show that reduced macrophage heterogeneity due to delayed emergence of restorative subsets underlies dysfunctional tissue repair during chronic infection. Our findings highlight how the preexisting inflammatory environment within tissue alters reparative immunity and ultimately the quality of tissue regeneration.

Therapeutic administration of IL-10 and amphiregulin alleviates chronic skeletal muscle inflammation and damage induced by infection.

Maintenance of tissue integrity in skeletal muscle requires the immunomodulatory and regenerative functions of muscle-resident regulatory T cells (Tregs). Chronic skeletal muscle infections, such as with Toxoplasma gondii disrupt normal immuno-regulatory networks and lead to pathogenic changes in Treg function. Specifically, Tregs during chronic T. gondii infection reinforce an inflammatory macrophage bias that exacerbates injury in skeletal muscle. In this study, we investigated whether the aberrations in skeletal muscle Treg function during chronic infection could be overcome by treatment with Treg-related factors associated with enhanced muscle regeneration during sterile injury. We show treatment of chronically infected mice with the Treg promoting therapies, interleukin-2 complexed with anti-IL-2 antibody or interleukin-33 (IL-33), did not restore macrophage dynamics or muscle function, respectively, in vivo. However supplementation of known Treg-derived factors, interleukin-10 (IL-10) and amphiregulin (Areg) improved muscle function and skewed macrophages toward a restorative phenotype in the presence of chronic infection. These shifts in macrophage phenotype are coupled with enhanced physiologic parameters of regeneration. Together, these data suggest that while Treg-mediated immuno-regulation is compromised during chronic skeletal muscle infection, supplementation of canonical Treg-derived factors such as IL-10 and Areg can restore immunologic balance and enhance muscle repair.

Cytokine responsiveness of CD8(+) T cells is a reproducible biomarker for the clinical efficacy of dendritic cell vaccination in glioblastoma patients.

BACKGROUND: Immunotherapeutic approaches, such as dendritic cell (DC) vaccination, have emerged as promising strategies in the treatment of glioblastoma. Despite their promise, however, the absence of objective biomarkers and/or immunological monitoring techniques to assess the clinical efficacy of immunotherapy still remains a primary limitation. To address this, we sought to identify a functional biomarker for anti-tumor immune responsiveness associated with extended survival in glioblastoma patients undergoing DC vaccination. METHODS: 28 patients were enrolled and treated in two different Phase 1 DC vaccination clinical trials at UCLA. To assess the anti-tumor immune response elicited by therapy, we studied the functional responsiveness of pre- and post-vaccination peripheral blood lymphocytes (PBLs) to the immunostimulatory cytokines interferon-gamma (IFN-γ) and interleukin-2 (IL-2) in 21 of these patients for whom we had adequate material. Immune responsiveness was quantified by measuring downstream phosphorylation events of the transcription factors, STAT-1 and STAT-5, via phospho-specific flow cytometry. RESULTS: DC vaccination induced a significant decrease in the half-maximal concentration (EC-50) of IL-2 required to upregulate pSTAT-5 specifically in CD3(+)CD8(+) T lymphocytes (p < 0.045). Extended survival was also associated with an increased per cell phosphorylation of STAT-5 in cytotoxic T-cells following IL-2 stimulation when the median post/pre pSTAT-5 ratio was used to dichotomize the patients (p = 0.0015, log-rank survival; hazard ratio = 0.1834, p = 0.018). Patients whose survival was longer than two years had a significantly greater pSTAT-5 ratio (p = 0.015), but, contrary to our expectations, a significantly lower pSTAT-1 ratio (p = 0.038). CONCLUSIONS: Our results suggest that monitoring the pSTAT signaling changes in PBL may provide a functional immune monitoring measure predictive of clinical efficacy in DC-vaccinated patients.