Metabolic Alterations Contribute to Enhanced Inflammatory Cytokine Production in Irgm1-deficient Macrophages.

The immunity-related GTPases (IRGs) are a family of proteins that are induced by interferon (IFN)-γ and play pivotal roles in immune and inflammatory responses. IRGs ostensibly function as dynamin-like proteins that bind to intracellular membranes and promote remodeling and trafficking of those membranes. Prior studies have shown that loss of Irgm1 in mice leads to increased lethality to bacterial infections as well as enhanced inflammation to non-infectious stimuli; however, the mechanisms underlying these phenotypes are unclear. In the studies reported here, we found that uninfected Irgm1-deficient mice displayed high levels of serum cytokines typifying profound autoinflammation. Similar increases in cytokine production were also seen in cultured, IFN-γ-primed macrophages that lacked Irgm1. A series of metabolic studies indicated that the enhanced cytokine production was associated with marked metabolic changes in the Irgm1-deficient macrophages, including increased glycolysis and an accumulation of long chain acylcarnitines. Cells were exposed to the glycolytic inhibitor, 2-deoxyglucose, or fatty acid synthase inhibitors to perturb the metabolic alterations, which resulted in dampening of the excessive cytokine production. These results suggest that Irgm1 deficiency drives metabolic dysfunction in macrophages in a manner that is cell-autonomous and independent of infectious triggers. This may be a significant contributor to excessive inflammation seen in Irgm1-deficient mice in different contexts.

IRG and GBP host resistance factors target aberrant, "non-self" vacuoles characterized by the missing of "self" IRGM proteins.

Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with "non-self" PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on "self" organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of "self" IRGM proteins from these structures.

Irgm1-deficient mice exhibit Paneth cell abnormalities and increased susceptibility to acute intestinal inflammation.

Crohn's disease (CD) is a chronic, immune-mediated, inflammatory disorder of the intestine that has been linked to numerous susceptibility genes, including the immunity-related GTPase (IRG) M (IRGM). IRGs comprise a family of proteins known to confer resistance to intracellular infections through various mechanisms, including regulation of phagosome processing, cell motility, and autophagy. However, despite its association with CD, the role of IRGM and other IRGs in regulating intestinal inflammation is unclear. We investigated the involvement of Irgm1, an ortholog of IRGM, in the genesis of murine intestinal inflammation. After dextran sodium sulfate exposure, Irgm1-deficient [Irgm1 knockout (KO)] mice showed increased acute inflammation in the colon and ileum, with worsened clinical responses. Marked alterations of Paneth cell location and granule morphology were present in Irgm1 KO mice, even without dextran sodium sulfate exposure, and were associated with impaired mitophagy and autophagy in Irgm1 KO intestinal cells (including Paneth cells). This was manifested by frequent tubular and swollen mitochondria and increased LC3-positive autophagic structures. Interestingly, these LC3-positive structures often contained Paneth cell granules. These results suggest that Irgm1 modulates acute inflammatory responses in the mouse intestine, putatively through the regulation of gut autophagic processes, that may be pivotal for proper Paneth cell functioning.

Immunity-related GTPase M (IRGM) proteins influence the localization of guanylate-binding protein 2 (GBP2) by modulating macroautophagy.

The immunity-related GTPases (IRGs) are a family of proteins induced by interferon-γ that play a crucial role in innate resistance to intracellular pathogens. The M subfamily of IRG proteins (IRGM) plays a profound role in this context, in part because of the ability of its members to regulate the localization and expression of other IRG proteins. We present here evidence that IRGM proteins affect the localization of the guanylate-binding proteins (GBPs), a second family of interferon-induced GTP-binding proteins that also function in innate immunity. Absence of Irgm1 or Irgm3 led to accumulation of Gbp2 in intracellular compartments that were positive for both the macroautophagy (hereafter referred to as autophagy) marker LC3 and the autophagic adapter molecule p62/Sqstm1. Gbp2 was similarly relocalized in cells in which autophagy was impaired because of the absence of Atg5. Both in Atg5- and IRGM-deficient cells, the IRG protein Irga6 relocalized to the same compartments as Gbp2, raising the possibility of a common regulatory mechanism. However, other data indicated that Irga6, but not Gbp2, was ubiquitinated in IRGM-deficient cells. Similarly, coimmunoprecipitation studies indicated that although Irgm3 did interact directly with Irgb6, it did not interact with Gbp2. Collectively, these data suggest that IRGM proteins indirectly modulate the localization of GBPs through a distinct mechanism from that through which they regulate IRG protein localization. Further, these results suggest that a core function of IRGM proteins is to regulate autophagic flux, which influences the localization of GBPs and possibly other factors that instruct cell-autonomous immune resistance.

Dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon alpha/beta.

Differentiation of dendritic cells (DCs) into particular subsets may act to shape innate and adaptive immune responses, but little is known about how this occurs during infections. Plasmacytoid dendritic cells (PDCs) are major producers of interferon (IFN)-alpha/beta in response to many viruses. Here, the functions of these and other splenic DC subsets are further analyzed after in vivo infection with murine cytomegalovirus (MCMV). Viral challenge induced PDC maturation, their production of high levels of innate cytokines, and their ability to activate natural killer (NK) cells. The conditions also licensed PDCs to efficiently activate CD8 T cells in vitro. Non-plasmacytoid DCs induced T lymphocyte activation in vitro. As MCMV preferentially infected CD8alpha+ DCs, however, restricted access to antigens may limit plasmacytoid and CD11b+ DC contribution to CD8 T cell activation. IFN-alpha/beta regulated multiple DC responses, limiting viral replication in all DC and IL-12 production especially in the CD11b+ subset but promoting PDC accumulation and CD8alpha+ DC maturation. Thus, during defense against a viral infection, PDCs appear specialized for initiation of innate, and as a result of their production of IFN-alpha/beta, regulate other DCs for induction of adaptive immunity. Therefore, they may orchestrate the DC subsets to shape endogenous immune responses to viruses.

Palmitoylation of the immunity related GTPase, Irgm1: impact on membrane localization and ability to promote mitochondrial fission.

The Immunity-Related GTPases (IRG) are a family of large GTPases that mediate innate immune responses. Irgm1 is particularly critical for immunity to bacteria and protozoa, and for inflammatory homeostasis in the intestine. Although precise functions for Irgm1 have not been identified, prior studies have suggested roles in autophagy/mitophagy, phagosome remodeling, cell motility, and regulating the activity of other IRG proteins. These functions ostensibly hinge on the ability of Irgm1 to localize to intracellular membranes, such as those of the Golgi apparatus and mitochondria. Previously, it has been shown that an amphipathic helix, the αK helix, in the C-terminal portion of the protein partially mediates membrane binding. However, in absence of αK, there is still substantial binding of Irgm1 to cellular membranes, suggesting the presence of other membrane binding motifs. In the current work, an additional membrane localization motif was found in the form of palmitoylation at a cluster of cysteines near the αK. An Irgm1 mutant possessing alanine to cysteine substitutions at these amino acids demonstrated little residual palmitoylation, yet it displayed only a small decrease in localization to the Golgi and mitochondria. In contrast, a mutant containing the palmitoylation mutations in combination with mutations disrupting the amphipathic character of the αK displayed a complete loss of apparent localization to the Golgi and mitochondria, as well as an overall loss of association with cellular membranes in general. Additionally, Irgm1 was found to promote mitochondrial fission, and this function was undermined in Irgm1 mutants lacking the palmitoylation domain, and to a greater extent in those lacking the αK, or the αK and palmitoylation domains combined. Our data suggest that palmitoylation together with the αK helix firmly anchor Irgm1 in the Golgi and mitochondria, thus facilitating function of the protein.

Regulation of macrophage motility by Irgm1.

IRG are a family of IFN-regulated proteins that are critical for resistance to infection. Mouse IRG proteins are divided into GMS and GKS subfamilies, based on a sequence within the G1 GTP-binding motif. The GMS proteins have a particularly profound impact on immunity, as typified by Irgm1, of which absence leads to a complete loss of resistance to a variety of intracellular bacteria and protozoa. The underlying molecular and cellular mechanisms are not clear. Here, we use time-lapse microscopy and cell-tracking analysis to demonstrate that Irgm1 is required for motility of IFN-gamma-activated macrophages. The absence of Irgm1 led to decreased actin remodeling at the leading edge of migrating macrophages, as well as decreased Rac activation. Although Irgm1 did not localize to the leading edge of migrating macrophages, it was found to regulate the localization of a GKS IRG protein, Irgb6, which in turn, concentrated on the plasma membrane in the advancing lamellipodia, in close apposition to molecular components that regulate membrane remodeling, including Rac, paxillin, and actin. Thus, Irgm1 likely controls macrophage motility by regulating the positioning of specific GKS IRG proteins to the plasma membrane, which in turn, modulate cytoskeletal remodeling and membrane dynamics.

Balance of Irgm protein activities determines IFN-gamma-induced host defense.

The immunity-related GTPases (IRG), also known as p47 GTPases, are a family of proteins that are tightly regulated by IFNs at the transcriptional level and serve as key mediators of IFN-regulated resistance to intracellular bacteria and protozoa. Among the IRG proteins, loss of Irgm1 has the most profound impact on IFN-gamma-induced host resistance at the physiological level. Surprisingly, the losses of host resistance seen in the absence of Irgm1 are sometimes more striking than those seen in the absence of IFN-gamma. In the current work, we address the underlying mechanism. We find that in several contexts, another protein in the IRG family, Irgm3, functions to counter the effects of Irgm1. By creating mice that lack Irgm1 and Irgm3, we show that several phenotypes important to host resistance that are caused by Irgm1 deficiency are reversed by coincident Irgm3 deficiency; these include resistance to Salmonella typhimurium in vivo, the ability to affect IFN-gamma-induced Salmonella killing in isolated macrophages, and the ability to regulate macrophage adhesion and motility in vitro. Other phenotypes that are caused by Irgm1 deficiency, including susceptibility to Toxoplasma gondii and the regulation of GKS IRG protein expression and localization, are not reversed but exacerbated when Irgm3 is also absent. These data suggest that members of the Irgm subfamily within the larger IRG family possess activities that can be opposing or cooperative depending on the context, and it is the balance of these activities that is pivotal in mediating IFN-gamma-regulated host resistance.

Behavioral characterization of P311 knockout mice.

P311 is an 8-kDa protein that is expressed in many brain regions, particularly the hippocampus, cerebellum and olfactory lobes, and is under stringent regulation by developmental, mitogenic and other physiological stimuli. P311 is thought to be involved in the transformation and motility of neural cells; however, its role in normal brain physiology is undefined. To address this point, P311-deficient mice were developed through gene targeting and their behaviors were characterized. Mutants displayed no overt abnormalities, bred normally and had normal survival rates. Additionally, no deficiencies were noted in motor co-ordination, balance, hearing or olfactory discrimination. Nevertheless, P311-deficient mice showed altered behavioral responses in learning and memory. These included impaired responses in social transmission of food preference, Morris water maze and contextual fear conditioning. Additionally, mutants displayed altered emotional responses as indicated by decreased freezing in contextual and cued fear conditioning and reduced fear-potentiated startle. Together, these data establish P311 as playing an important role in learning and memory processes and emotional responses.

Impaired macrophage function underscores susceptibility to Salmonella in mice lacking Irgm1 (LRG-47).

IRG proteins, or immunity-related GTPases (also known as p47 GTPases), are a group of IFN-regulated proteins that are highly expressed in response to infection. The proteins localize to intracellular membranes including vacuoles that contain pathogens in infected macrophages and other host cells. Current data indicate that the IRG protein Irgm1 (LRG-47) is critical for resistance to intracellular bacteria. This function is thought to be a consequence of regulating the survival of vacuolar bacteria in host cells. In the current work, the role of Irgm1 in controlling resistance to Salmonella typhimurium was explored to further define the mechanism through which the protein regulates host resistance. Irgm1-deficient mice displayed increased susceptibility to this bacterium that was reflected in increased bacterial loads in spleen and liver and decreased maturation of S. typhimurium granulomas. The mice also displayed an inability to concentrate macrophages at sites of bacterial deposition. In vitro, the ability of Irgm1-deficient macrophages to suppress intracellular growth of S. typhimurium was impaired. Furthermore, adhesion and motility of Irgm1-deficient macrophages after activation with IFN-gamma was markedly decreased. Altered adhesion/motility of those cells was accompanied by changes in cell morphology, density of adhesion-associated proteins, and actin staining. Together, these data suggest that in addition to regulating the maturation of pathogen-containing vacuoles, Irgm1 plays a key role in regulating the adhesion and motility of activated macrophages.

Nasal peptide vaccination elicits CD8 responses and reduces viral burden after challenge with virulent murine cytomegalovirus.

Infection of BALB/c mice with murine cytomegalovirus (MCMV) leads to CD8 cell responses to an immunodominant epitope YPHFMPTNL. We presented this epitope as a nasal peptide vaccine in combination with cholera toxin adjuvant, and evaluated immune responses and protection from MCMV challenge. Vaccination of naive mice generated elevated numbers of peptide-specific interferon-gamma-secreting splenocytes (median 80/million, range 60 to 490), compared to control mice (median 2/million, range -4.5 to 8; P=0.008, Mann-Whitney test). Twelve days after challenge with virulent MCMV, vaccinated mice had a 1.1 log(10) reduction in salivary gland viral titer compared to unvaccinated controls (5.36+/-0.24 vs. 6.42+/-0.12, mean +/-SD log(10) plaque-forming-units; P <0.001, t -test). Mice with chronic MCMV infection had consistent responses to the peptide (183+/-24/million interferon-gamma-secreting splenocytes). Nasal peptide vaccination during chronic infection boosted peptide-specific responses in two of four mice to >900/million interferon-gamma-secreting splenocytes. Nasal peptide vaccination was immunogenic in naïve and MCMV-infected mice, and reduced viral burden in naive mice after virulent MCMV challenge. The nasal route may be useful for peptide presentation by novel human vaccines.

p47 GTPases regulate Toxoplasma gondii survival in activated macrophages.

The cytokine gamma interferon (IFN-gamma) is critical for resistance to Toxoplasma gondii. IFN-gamma strongly activates macrophages and nonphagocytic host cells to limit intracellular growth of T. gondii; however, the cellular factors that are required for this effect are largely unknown. We have shown previously that IGTP and LRG-47, members of the IFN-gamma-regulated family of p47 GTPases, are required for resistance to acute T. gondii infections in vivo. In contrast, IRG-47, another member of this family, is not required. In the present work, we addressed whether these GTPases are required for IFN-gamma-induced suppression of T. gondii growth in macrophages in vitro. Bone marrow macrophages that lacked IGTP or LRG-47 displayed greatly attenuated IFN-gamma-induced inhibition of T. gondii growth, while macrophages that lacked IRG-47 displayed normal inhibition. Thus, the ability of the p47 GTPases to limit acute infection in vivo correlated with their ability to suppress intracellular growth in macrophages in vitro. Using confocal microscopy and sucrose density fractionation, we demonstrated that IGTP largely colocalizes with endoplasmic reticulum markers, while LRG-47 was mainly restricted to the Golgi. Although both IGTP and LRG-47 localized to vacuoles containing latex beads, neither protein localized to vacuoles containing live T. gondii. These results suggest that IGTP and LRG-47 are able to regulate host resistance to acute T. gondii infections through their ability to inhibit parasite growth within the macrophage.

A lymphotoxin-IFN-beta axis essential for lymphocyte survival revealed during cytomegalovirus infection.

The importance of lymphotoxin (LT) betaR (LTbetaR) as a regulator of lymphoid organogenesis is well established, but its role in host defense has yet to be fully defined. In this study, we report that mice deficient in LTbetaR signaling were highly susceptible to infection with murine CMV (MCMV) and early during infection exhibited a catastrophic loss of T and B lymphocytes, although the majority of lymphocytes were themselves not directly infected. Moreover, bone marrow chimeras revealed that lymphocyte survival required LTalpha expression by hemopoietic cells, independent of developmental defects in lymphoid tissue, whereas LTbetaR expression by both stromal and hemopoietic cells was needed to prevent apoptosis. The induction of IFN-beta was also severely impaired in MCMV-infected LTalpha(-/-) mice, but immunotherapy with an agonist LTbetaR Ab restored IFN-beta levels, prevented lymphocyte death, and enhanced the survival of these mice. IFN-alphabetaR(-/-) mice were also found to exhibit profound lymphocyte death during MCMV infection, thus providing a potential mechanistic link between type 1 IFN induction and lymphocyte survival through a LTalphabeta-dependent pathway important for MCMV host defense.

In vivo protein expression and immune responses generated by DNA vaccines expressing mycobacterial antigens fused with a reporter protein.

We cloned six mycobacterial antigens into a mammalian expression vector as fusion proteins with the enhanced green fluorescent protein (EGFP). Plasmid DNA was injected intramuscularly, and the injection sites were examined 1 week later. Expression of each antigen-EGFP fusion protein was visualized as green fluorescence in muscle tissue sections. A plasmid expressing EGFP alone and a plasmid with a frameshift mutation served as positive and negative controls. Visualization of fluorescent protein in vivo was 100% specific when compared to in vitro results. In vivo sensitivity was only 37% based on individual injection sites, but increased to 100% when results from multiple injection sites were combined for each plasmid. EGFP alone was expressed in a higher proportion of myocytes than the antigen-EGFP fusion proteins (P < 0.001). There was a trend toward an inverse correlation between protein size and the proportion of myocytes with visible fluorescence (r = -0.68; P = 0.09). We compared antibody subtypes generated to Mycobacterium bovis antigen 85A, when it was expressed alone or as a fusion protein. Inclusion of EGFP modified the immune response toward a Th1 response, as indicated by the ratio of antigen 85A-specific IgG2a to IgG1 generated by each plasmid (antigen 85A alone 0.73 +/- 0.18 versus antigen 85A-EGFP 1.82 +/- 0.57, mean +/- S.D.; P < 0.01), though the magnitude of the antibody isotype shift was modest. Direct visualization of antigen-EGFP fusion proteins provided a simple and rapid method to confirm in vivo antigen expression.