The Toxoplasma oxygen-sensing protein, TgPhyA, is required for resistance to interferon gamma-mediated nutritional immunity in mice.

As Toxoplasma gondii disseminates through its host, the parasite must sense and adapt to its environment and scavenge nutrients. Oxygen (O2) is one such environmental factor and cytoplasmic prolyl 4-hydroxylases (PHDs) are evolutionarily conserved O2 cellular sensing proteins that regulate responses to changes in O2 availability. Toxoplasma expresses 2 PHDs. One of them, TgPHYa hydroxylates SKP1, a subunit of the SCF-E3 ubiquitin ligase complex. In vitro, TgPHYa is important for growth at low O2 levels. However, studies have yet to examine the role that TgPHYa or any other pathogen-encoded PHD plays in virulence and disease. Using a type II ME49 Toxoplasma TgPHYa knockout, we report that TgPHYa is important for Toxoplasma virulence and brain cyst formation in mice. We further find that while TgPHYa mutant parasites can establish an infection in the gut, they are unable to efficiently disseminate to peripheral tissues because the mutant parasites are unable to survive within recruited immune cells. Since this phenotype was abrogated in IFNγ knockout mice, we studied how TgPHYa mediates survival in IFNγ-treated cells. We find that TgPHYa is not required for release of parasite-encoded effectors into host cells that neutralize anti-parasitic processes induced by IFNγ. In contrast, we find that TgPHYa is required for the parasite to scavenge tryptophan, which is an amino acid whose levels are decreased after IFNγ up-regulates the tryptophan-catabolizing enzyme, indoleamine dioxygenase (IDO). We further find, relative to wild-type mice, that IDO knockout mice display increased morbidity when infected with TgPHYa knockout parasites. Together, these data identify the first parasite mechanism for evading IFNγ-induced nutritional immunity and highlight a novel role that oxygen-sensing proteins play in pathogen growth and virulence.

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.

Rational design of a nanoparticle platform for oral prophylactic immunotherapy to prevent immunogenicity of therapeutic proteins.

The safety and efficacy of several life-saving therapeutic proteins are compromised due to their immunogenicity. Once a sustained immune response against a protein-based therapy is established, clinical options that are safe and cost-effective become limited. Prevention of immunogenicity of therapeutic proteins prior to their initial use is critical as it is often difficult to reverse an established immune response. Here, we discuss a rational design and testing of a phosphatidylserine-containing nanoparticle platform for novel oral prophylactic reverse vaccination approach, i.e., pre-treatment of a therapeutic protein in the presence of nanoparticles to prevent immunogenicity of protein therapies.

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.

Cryptococcus neoformans Coinfection Dampens the TNF-α Response in HIV-1-Infected Human THP-1 Macrophages.

Cryptococcus neoformans is a devastating opportunistic fungal pathogen. It mostly impacts people in an immunocompromised state, such as people living with HIV/AIDS and following organ transplantation. Macrophages, in addition to being a major cellular reservoir of HIV-1, represent a unique niche in which both C. neoformans and HIV-1 can coinhabit in the course of natural infection. Here, we report the observation that HIV-1 infection of THP-1 macrophages increases the rate at which they phagocytose C. neoformans cells. We investigated the tumor necrosis factor alpha (TNF-α) signaling and nuclear factor kappa B (NF-κB) activation in human monocyte-derived macrophages infected with HIV-1 alone, as well as those coinfected with HIV-1 and C. neoformans Our findings showed that while HIV-1 infection alone upregulates TNF-α production and activates NF-κB signaling, C. neoformans coinfection drastically and rapidly dampens this proinflammatory response. These data suggest an antagonism between two important human pathogens during coinfection of macrophages.IMPORTANCE Fungal infections are one of the leading causes of death for people who live with HIV/AIDS. Even though these pathogens are independently well studied, it is still enigmatic how coinfection with HIV-1 and C. neoformans alters gene expression and cellular processes, especially in clinically relevant cell types. Understanding the interplay between these two pathogens is especially critical because C. neoformans mortality largely depends on the host's immunocompromised state during viral infection. Studying this coinfection is challenging since HIV-1 only infects human cells, and the modified murine HIV-1 virus does not reproduce the clinical landmarks of HIV-1 infection or AIDS in mice. Our observations shed light on how these two pathogens trigger opposing trends in TNF-α and NF-κB signaling in human monocyte-derived macrophages.

SARS-CoV-2 RBD Neutralizing Antibody Induction is Enhanced by Particulate Vaccination.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a candidate vaccine antigen that binds angiotensin-converting enzyme 2 (ACE2), leading to virus entry. Here, it is shown that rapid conversion of recombinant RBD into particulate form via admixing with liposomes containing cobalt-porphyrin-phospholipid (CoPoP) potently enhances the functional antibody response. Antigen binding via His-tag insertion into the CoPoP bilayer results in a serum-stable and conformationally intact display of the RBD on the liposome surface. Compared to other vaccine formulations, immunization using CoPoP liposomes admixed with recombinant RBD induces multiple orders of magnitude higher levels of antibody titers in mice that neutralize pseudovirus cell entry, block RBD interaction with ACE2, and inhibit live virus replication. Enhanced immunogenicity can be accounted for by greater RBD uptake into antigen-presenting cells in particulate form and improved immune cell infiltration in draining lymph nodes. QS-21 inclusion in the liposomes results in an enhanced antigen-specific polyfunctional T cell response. In mice, high dose immunization results in minimal local reactogenicity, is well-tolerated, and does not elevate serum cobalt levels. Taken together, these results confirm that particulate presentation strategies for the RBD immunogen should be considered for inducing strongly neutralizing antibody responses against SARS-CoV-2.

Neutrophils Are Required During Immunization With the Pneumococcal Conjugate Vaccine for Protective Antibody Responses and Host Defense Against Infection.

Neutrophils can shape adaptive immunity; however, their role in vaccine-induced protection against infections in vivo remains unclear. Here, we tested their role in the clinically relevant polysaccharide conjugate vaccine against Streptococcus pneumoniae (pneumococcus). We antibody depleted neutrophils during vaccination, allowed them to recover, and 4 weeks later challenged mice with pneumococci. We found that while isotype-treated vaccinated controls were protected against an otherwise lethal infection in naive mice, full protection was lost upon neutrophil depletion. Compared to vaccinated controls, neutrophil-depleted mice had higher lung bacterial burdens, increased incidence of bacteremia, and lower survival rates. Sera from neutrophil-depleted mice had less antipneumococcal IgG2c and IgG3, were less efficient at inducing opsonophagocytic killing of bacteria by neutrophils in vitro, and were worse at protecting naive mice against pneumococcal pneumonia. In summary, neutrophils are required during vaccination for optimal host protection, which has important implications for future vaccine design against pneumococci and other pathogens.

Toxoplasma infection induces microglia-neuron contact and the loss of perisomatic inhibitory synapses.

Infection and inflammation within the brain induces changes in neuronal connectivity and function. The intracellular protozoan parasite, Toxoplasma gondii, is one pathogen that infects the brain and can cause encephalitis and seizures. Persistent infection by this parasite is also associated with behavioral alterations and an increased risk for developing psychiatric illness, including schizophrenia. Current evidence from studies in humans and mouse models suggest that both seizures and schizophrenia result from a loss or dysfunction of inhibitory synapses. In line with this, we recently reported that persistent T. gondii infection alters the distribution of glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes GABA synthesis in inhibitory synapses. These changes could reflect a redistribution of presynaptic machinery in inhibitory neurons or a loss of inhibitory nerve terminals. To directly assess the latter possibility, we employed serial block face scanning electron microscopy (SBFSEM) and quantified inhibitory perisomatic synapses in neocortex and hippocampus following parasitic infection. Not only did persistent infection lead to a significant loss of perisomatic synapses, it induced the ensheathment of neuronal somata by myeloid-derived cells. Immunohistochemical, genetic, and ultrastructural analyses revealed that these myeloid-derived cells included activated microglia. Finally, ultrastructural analysis identified myeloid-derived cells enveloping perisomatic nerve terminals, suggesting they may actively displace or phagocytose synaptic elements. Thus, these results suggest that activated microglia contribute to perisomatic inhibitory synapse loss following parasitic infection and offer a novel mechanism as to how persistent T. gondii infection may contribute to both seizures and psychiatric illness.

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.

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.

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.

Brains and Brawn: Toxoplasma Infections of the Central Nervous System and Skeletal Muscle.

Toxoplasma gondii is a widespread parasitic pathogen that infects over a third of the world's population. Following an acute infection, the parasite can persist within its mammalian host as intraneuronal or intramuscular cysts. Cysts will occasionally reactivate, and - depending on the host's immune status and site of reactivation - encephalitis or myositis can develop. Because these diseases have high levels of morbidity and can be lethal, it is important to understand how Toxoplasma traffics to these tissues, how the immune response controls parasite burden and contributes to tissue damage, and what mechanisms underlie neurological and muscular pathologies that toxoplasmosis patients present with. This review aims to summarize recent important developments addressing these critical topics.

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.

In Vitro Analyses of T Cell Effector Differentiation.

In vitro culture is an important complement, or substitute, to in vivo approaches in order to study T cell effector differentiation. Here, we describe culture conditions that generate specific effector cell types by exposing naïve T cells to appropriate cytokine signals.

Cbl-b Deficiency in Mice Results in Exacerbation of Acute and Chronic Stages of Allergic Asthma.

Mice sensitized to ovalbumin (OVA) develop allergic airway disease (AAD) with short-term daily OVA aerosol challenge; inflammation resolves with long-term OVA aerosol exposure, resulting in local inhalational tolerance (LIT). Cbl-b is an E3 ubiquitin ligase involved with CD28 signaling; Cbl-b(-/-) effector T cells are resistant to regulatory T cell-mediated suppression in vitro and in vivo. The present study utilized Cbl-b(-/-) mice to investigate the role of Cbl-b in the development of AAD and LIT. Cbl-b(-/-) mice exhibited increased airway inflammation during AAD, which failed to resolve with long-term OVA aerosol exposure. Exacerbation of inflammation in Cbl-b(-/-) mice correlated with increased proinflammatory cytokine levels and expansion of effector T cells in the BAL during AAD, but did not result in either a modulation of lymphocyte subsets in systemic tissues or in OVA-specific IgE in serum. These results implicate a role for Cbl-b in the resolution of allergic airway inflammation.

The alarmin IL-33 promotes regulatory T-cell function in the intestine.

FOXP3(+) regulatory T cells (Treg cells) are abundant in the intestine, where they prevent dysregulated inflammatory responses to self and environmental stimuli. It is now appreciated that Treg cells acquire tissue-specific adaptations that facilitate their survival and function; however, key host factors controlling the Treg response in the intestine are poorly understood. The interleukin (IL)-1 family member IL-33 is constitutively expressed in epithelial cells at barrier sites, where it functions as an endogenous danger signal, or alarmin, in response to tissue damage. Recent studies in humans have described high levels of IL-33 in inflamed lesions of inflammatory bowel disease patients, suggesting a role for this cytokine in disease pathogenesis. In the intestine, both protective and pathological roles for IL-33 have been described in murine models of acute colitis, but its contribution to chronic inflammation remains ill defined. Here we show in mice that the IL-33 receptor ST2 is preferentially expressed on colonic Treg cells, where it promotes Treg function and adaptation to the inflammatory environment. IL-33 signalling in T cells stimulates Treg responses in several ways. First, it enhances transforming growth factor (TGF)-ß1-mediated differentiation of Treg cells and, second, it provides a necessary signal for Treg-cell accumulation and maintenance in inflamed tissues. Strikingly, IL-23, a key pro-inflammatory cytokine in the pathogenesis of inflammatory bowel disease, restrained Treg responses through inhibition of IL-33 responsiveness. These results demonstrate a hitherto unrecognized link between an endogenous mediator of tissue damage and a major anti-inflammatory pathway, and suggest that the balance between IL-33 and IL-23 may be a key controller of intestinal immune responses.

A mouse model of HIES reveals pro- and anti-inflammatory functions of STAT3.

Mutations of STAT3 underlie the autosomal dominant form of hyperimmunoglobulin E syndrome (HIES). STAT3 has critical roles in immune cells and thus, hematopoietic stem cell transplantation (HSCT), might be a reasonable therapeutic strategy in this disease. However, STAT3 also has critical functions in nonhematopoietic cells and dissecting the protean roles of STAT3 is limited by the lethality associated with germline deletion of Stat3. Thus, predicting the efficacy of HSCT for HIES is difficult. To begin to dissect the importance of STAT3 in hematopoietic and nonhematopoietic cells as it relates to HIES, we generated a mouse model of this disease. We found that these transgenic mice recapitulate multiple aspects of HIES, including elevated serum IgE and failure to generate Th17 cells. We found that these mice were susceptible to bacterial infection that was partially corrected by HSCT using wild-type bone marrow, emphasizing the role played by the epithelium in the pathophysiology of HIES.

Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells.

A proper balance between Th17 and T regulatory cells (Treg cells) is critical for generating protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk (IL2-inducible T cell kinase), a component of T cell receptor (TCR) signaling pathways, influences this balance by regulating cross talk between TCR and cytokine signaling. Under both Th17 and Treg cell differentiation conditions, Itk(-/-) CD4(+) T cells develop higher percentages of functional FoxP3(+) cells, associated with increased sensitivity to IL-2. Itk(-/-) CD4(+) T cells also preferentially develop into Treg cells in vivo. We find that Itk-deficient T cells exhibit reduced TCR-induced phosphorylation of mammalian target of rapamycin (mTOR) targets, accompanied by downstream metabolic alterations. Surprisingly, Itk(-/-) cells also exhibit reduced IL-2-induced mTOR activation, despite increased STAT5 phosphorylation. We demonstrate that in wild-type CD4(+) T cells, TCR stimulation leads to a dose-dependent repression of Pten. However, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and phosphoinositide-3-kinase (PI3K) pathways. Moreover, Itk-deficient CD4(+) T cells show impaired TCR-mediated induction of Myc and miR-19b, known repressors of Pten. Our results demonstrate that Itk helps orchestrate positive feedback loops integrating multiple T cell signaling pathways, suggesting Itk as a potential target for altering the balance between Th17 and Treg cells.

Inflammatory monocytes regulate pathologic responses to commensals during acute gastrointestinal infection.

The commensal flora can promote both immunity to pathogens and mucosal inflammation. How commensal-driven inflammation is regulated in the context of infection remains poorly understood. Here, we show that during acute mucosal infection of mice with Toxoplasma gondii, inflammatory monocytes acquire a tissue-specific regulatory phenotype associated with production of the lipid mediator prostaglandin E2 (PGE2). Notably, in response to commensals, inflammatory monocytes can directly inhibit neutrophil activation in a PGE2-dependent manner. Further, in the absence of inflammatory monocytes, mice develop severe neutrophil-mediated pathology in response to pathogen challenge that can be controlled by PGE2 analog treatment. Complementing these findings, inhibition of PGE2 led to enhanced neutrophil activation and host mortality after infection. These data demonstrate a previously unappreciated dual action of inflammatory monocytes in controlling pathogen expansion while limiting commensal-mediated damage to the gut. Collectively, our results place inflammatory monocyte-derived PGE2 at the center of a commensal-driven regulatory loop required to control host-commensal dialog during pathogen-induced inflammation.

GATA3 controls Foxp3⁺ regulatory T cell fate during inflammation in mice.

Tregs not only keep immune responses to autoantigens in check, but also restrain those directed toward pathogens and the commensal microbiota. Control of peripheral immune homeostasis by Tregs relies on their capacity to accumulate at inflamed sites and appropriately adapt to their local environment. To date, the factors involved in the control of these aspects of Treg physiology remain poorly understood. Here, we show that the canonical Th2 transcription factor GATA3 is selectively expressed in Tregs residing in barrier sites including the gastrointestinal tract and the skin. GATA3 expression in both murine and human Tregs was induced upon TCR and IL-2 stimulation. Although GATA3 was not required to sustain Treg homeostasis and function at steady state, GATA3 played a cardinal role in Treg physiology during inflammation. Indeed, the intrinsic expression of GATA3 by Tregs was required for their ability to accumulate at inflamed sites and to maintain high levels of Foxp3 expression in various polarized or inflammatory settings. Furthermore, our data indicate that GATA3 limits Treg polarization toward an effector T cell phenotype and acquisition of effector cytokines in inflamed tissues. Overall, our work reveals what we believe to be a new facet in the complex role of GATA3 in T cells and highlights what may be a fundamental role in controlling Treg physiology during inflammation.