The immunity-related GTPase Irgm1 promotes the expansion of activated CD4+ T cell populations by preventing interferon-gamma-induced cell death.

Mice deficient in the interferon-gamma (IFN-gamma)-inducible, immunity-related GTPase Irgm1 have defective host resistance to a variety of intracellular pathogens. This greater susceptibility to infection is associated with impaired IFN-gamma-dependent macrophage microbicidal activity in vitro. Here we show that Irgm1 also regulated the survival of mature effector CD4(+) T lymphocytes by protecting them from IFN-gamma-induced autophagic cell death. Mice deficient in both IFN-gamma and Irgm1 were 'rescued' from the lymphocyte depletion and greater mortality that occurs in mice singly deficient in Irgm1 after mycobacterial infection. Our studies identify a feedback mechanism in the T helper type 1 response that limits the detrimental effects of IFN-gamma on effector T lymphocyte survival while promoting the antimicrobial functions of IFN-gamma.

Innate and adaptive interferons suppress IL-1α and IL-1ß production by distinct pulmonary myeloid subsets during Mycobacterium tuberculosis infection.

Interleukin-1 (IL-1) receptor signaling is necessary for control of Mycobacterium tuberculosis (Mtb) infection, yet the role of its two ligands, IL-1α and IL-1ß, and their regulation in vivo are poorly understood. Here, we showed that both IL-1α and IL-1ß are critically required for host resistance and identified two multifunctional inflammatory monocyte-macrophage and DC populations that coexpressed both IL-1 species at the single-cell level in lungs of Mtb-infected mice. Moreover, we demonstrated that interferons (IFNs) played important roles in regulating IL-1 production by these cells in vivo. Type I interferons inhibited IL-1 production by both subsets whereas CD4(+) T cell-derived IFN-γ selectively suppressed monocyte-macrophages. These data provide a cellular basis for both the anti-inflammatory effects of IFNs and probacterial functions of type I IFNs during Mtb infection and reveal differential regulation of IL-1 production by distinct cell populations as an additional layer of complexity in the activity of IL-1 in vivo.

Pillars article: downregulation of Th1 cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni. J. Exp. Med. 1991. 173: 159-166.

In the mouse, infection with Schistosoma mansoni results in an egg-producing infection and associated disease, whereas vaccination with attenuated larval stages produces a substantial and specific immunity in the absence of egg-induced pathology. Preliminary data showing enhanced interleukin-5 (IL-5) production by T cells from infected mice and interferon γ (IFN-γ) synthesis by cells from vaccinated animals (7), suggested differential CD4(+) subset stimulation by the different parasite stimuli. To confirem this hyposthesis, lymphocytes from vaccinated or infected animals were compared for their ability to produce IFN-γ and IL-2 (secreted by Th1 cells) as compared with IL-4 and IL-5 (characteristic Th2 cytokines). After stimulation with specific antigen or mitogen, T cells from vaccinated mice or prepatently infected animals responded primarily with Th1 lymphokines, whereas lymphocytes from patenly infected mice instead produced Th2 cytokines. The Th2 response in infected animals was shown to be induced by schistosome eggs and directed largely against egg antigens, whereas the Th1 reactivity in vaccinated mice was triggered primarily by larval anigens. Interestingly, Th1 responses in mice carrying egg-producing infections were found to be profoundly downregulated. Moreover, the injection of eggs into vaccinated mice resulted in a reduction of antigen and mitogen-stimulated Th1 function accompanied by a coincident expression of Th2 responses. Together, the data suggest that coincident with the induction of Th2 responses, murine schistosome infection results in an inhibition of potentially protective Th1 function. This previously unrecognized downregulation of Th1 cytokine production may be an important immunological consequence of helminth infection related to host adaptation.

Th1-driven immune reconstitution disease in Mycobacterium avium-infected mice.

Following antiretroviral therapy, a significant proportion of HIV(+) patients with mycobacterial coinfections develop a paradoxical, poorly understood inflammatory disease termed immune reconstitution inflammatory syndrome (IRIS). Here, we show that Mycobacterium avium-infected T cell-deficient mice injected with CD4 T cells also develop an immune reconstitution disease (IRD) manifesting as weight loss, impaired lung function, and rapid mortality. This form of IRD requires Ag recognition and interferonγ production by the donor CD4 T cells and correlates with marked alterations in blood and tissue CD11b(+) myeloid cells. Interestingly, disease is associated with impaired, rather than augmented, T-cell expansion and function and is not strictly dependent on lymphopenia-induced T-cell proliferation. Instead, our findings suggest that mycobacterial-associated IRIS results from a heightened sensitivity of infected lymphopenic hosts to the detrimental effects of Ag-driven CD4 T-cell responses.

Caspase-1 independent IL-1beta production is critical for host resistance to mycobacterium tuberculosis and does not require TLR signaling in vivo.

To investigate the respective contributions of TLR versus IL-1R mediated signals in MyD88 dependent control of Mycobacterium tuberculosis, we compared the outcome of M. tuberculosis infection in MyD88, TRIF/MyD88, IL-1R1, and IL-1beta-deficient mice. All four strains displayed acute mortality with highly increased pulmonary bacterial burden suggesting a major role for IL-1beta signaling in determining the MyD88 dependent phenotype. Unexpectedly, the infected MyD88 and TRIF/MyD88-deficient mice, rather than being defective in IL-1beta expression, displayed increased cytokine levels relative to wild-type animals. Similarly, infected mice deficient in caspase-1 and ASC, which have critical functions in inflammasome-mediated IL-1beta maturation, showed unimpaired IL-1beta production and importantly, were considerably less susceptible to infection than IL-1beta deficient mice. Together our findings reveal a major role for IL-1beta in host resistance to M. tuberculosis and indicate that during this infection the cytokine can be generated by a mechanism that does not require TLR signaling or caspase-1.

Bacteria-triggered CD4(+) T regulatory cells suppress Helicobacter hepaticus-induced colitis.

We have previously demonstrated that interleukin (IL)-10-deficient (IL-10 knockout [KO]) but not wild-type (WT) mice develop colitis after infection with Helicobacter hepaticus. Here, we show that infected recombination activating gene (RAG) KO mice develop intestinal inflammation after reconstitution with CD4(+) T cells from IL-10 KO animals and that the cotransfer of CD4(+) T cells from H. hepaticus-infected but not uninfected WT mice prevents this colitis. The disease-protective WT CD4(+) cells are contained within the CD45RB(low) fraction and unexpectedly were found in both the CD25(+) and the CD25(-) subpopulations of these cells, their frequency being higher in the latter. The mechanism by which CD25(+) and CD25(-) CD45RB(low) CD4(+) cells block colitis involves IL-10 and not transforming growth factor (TGF)-beta, as treatment with anti-IL-10R but not anti-TGF-beta monoclonal antibody abrogated their protective effect. In vitro, CD45RB(low) CD4(+) cells from infected WT mice were shown to produce IL-10 and suppress interferon-gamma production by IL-10 KO CD4(+) cells in an H. hepaticus antigen-specific manner. Together, our data support the concept that H. hepaticus infection results in the induction in WT mice of regulatory T cells that prevent bacteria-induced colitis. The induction of such cells in response to gut flora may be a mechanism protecting normal individuals against inflammatory bowel disease.

Maintenance of pulmonary Th1 effector function in chronic tuberculosis requires persistent IL-12 production.

The mechanisms that prevent reactivation of latent Mycobacterium tuberculosis infection in asymptomatic individuals are poorly understood. Although IL-12 is critical for the induction of IFN-gamma-dependent host control of M. tuberculosis, the requirement for the cytokine in the maintenance of host resistance and pulmonary Th1 effector function has not yet been formally examined. In this study, we reconstituted IL-12p40-deficient mice with IL-12 during the first 4 wk of infection and then assessed the effects of cytokine withdrawal. Although IL-12 administration initially resulted in restricted mycobacterial growth and prolonged survival, the reconstituted animals eventually succumbed to infection. This breakdown in bacterial control was accompanied by a marked reduction in the numbers of IFN-gamma-producing CD4(+) T cells in lungs. Moreover, whereas CD4(+) T cells isolated from chronically infected wild-type mice expanded and transferred long-term protection to M. tuberculosis-challenged RAG(-/-) mice, they failed to do so in IL-12p40-deficient RAG(-/-) recipients and were clearly reduced in frequency within pulmonary granulomas in the latter animals. These studies establish that continuous IL-12 production is necessary for maintenance of the pulmonary Th1 cells required for host control of persistent M. tuberculosis infection and suggest that breakdown of this mechanism could be a contributing factor in reactivated disease.

Parasite-induced Th2 polarization is associated with down-regulated dendritic cell responsiveness to Th1 stimuli and a transient delay in T lymphocyte cycling.

The nature of the signals that bias Th effector choice is still not completely understood. Using parasite extracts from pathogens known to induce polarized Th1 or Th2 responses and an in vitro experimental model for priming murine CD4(+) cells, we demonstrated that splenic dendritic cells (DC), but not B cells, promote Th1/Th2 differentiation of naive CD4(+) lymphocytes. Th polarization in this system was found not to depend on DC secretion of the polarizing cytokines IL-12/IL-4, but instead correlated with distinct states of DC activation induced by the different parasite preparations. As expected, conditioning of DC for Th1 development was associated with up-regulation of costimulatory molecules and enhanced chemokine production and required intact MyD88 signaling. In contrast, conditioning of DC for Th2 differentiation correlated with down-regulation of many of the same functions and was MyD88 independent. This dampened DC activation was accompanied in the cocultures by a reduction in the frequency of CD4(+) lymphocytes exiting the first division of the cell cycle. When the latter was mimicked by drug-induced arrest of peptide-primed CD4(+) cells after the S phase of the first cycle, a marked Th2 polarization was also observed. Together, these findings suggest that the emergence of IL-4-producing CD4(+) lymphocytes results from a suppression in DC function leading to a temporary delay in initial T cell cycling.

In the absence of IL-12, CD4(+) T cell responses to intracellular pathogens fail to default to a Th2 pattern and are host protective in an IL-10(-/-) setting.

IL-12-deficient mice exposed to nonlethal infections with intracellular pathogens or repeatedly immunized with a pathogen extract developed lowered but nevertheless substantial numbers of IFN-gamma(+) CD4(+) T cells compared to those observed in wild-type animals. Moreover, the CD4(+) responses in these knockout animals failed to default to a Th2 pattern. The protective efficacy of the Th1 cells developing in an IL-12-deficient setting was found to be limited by IL-10 since mice doubly deficient in IL-10 and IL-12 survived, while animals deficient in IL-12 alone succumbed to pathogen challenge. In contrast to IL-12 knockout mice, MyD88-deficient animals exposed to a Th1 microbial stimulus developed a pure Th2 response, arguing that this signaling element plays a more critical function than IL-12 in determining pathogen-induced CD4 polarization.

Transgenic mice expressing human interleukin-10 in the antigen-presenting cell compartment show increased susceptibility to infection with Mycobacterium avium associated with decreased macrophage effector function and apoptosis.

Interleukin-10 (IL-10) is thought to play an important role in the regulation of microbial immunity. While T-cell-derived IL-10 has been shown to suppress cell-mediated immunity, there has been debate as to whether antigen presenting cell (APC)-derived cytokine can perform the same function in vivo. To assess the influence of APC-produced IL-10 on host resistance to mycobacterial infection, transgenic mice expressing human IL-10 under the control of the major histocompatibility complex class II promoter (hu10Tg) were infected with Mycobacterium avium, and bacterial burdens and immune responses were compared with those observed in wild-type (wt) animals. Hu10Tg mice harbored substantially higher numbers of M. avium and succumbed 16 to 18 weeks postinfection. The granulomas in infected hu10Tg mice showed marked increases in both acid-fast bacilli and host macrophages. In addition, these animals displayed a dramatic increase in hepatic fibrosis. The increased susceptibility of the hu10Tg mice to M. avium infection is independent of T-cell-produced endogenous murine IL-10, since bacterial burdens in mice derived by crossing hu10Tg mice with murine IL-10-deficient mice were not significantly different from those in hu10Tg mice. Importantly, gamma interferon (IFN-gamma) responses were not decreased in the infected transgenic animals from those in wt animals, suggesting the normal development of Th1 effector cells. In contrast, mycobacterium-induced macrophage apoptosis as well as production of TNF, nitric oxide, and IL-12p40 were strongly inhibited in hu10Tg mice. Together, these data indicate that APC-derived IL-10 can exert a major inhibitory effect on control of mycobacterial infection by a mechanism involving the suppression of macrophage effector function and apoptosis.

The function of gamma interferon-inducible GTP-binding protein IGTP in host resistance to Toxoplasma gondii is Stat1 dependent and requires expression in both hematopoietic and nonhematopoietic cellular compartments.

IGTP is a member of the 47-kDa family of gamma interferon (IFN-gamma)-induced GTPases. We have previously shown that IGTP is critical for host resistance to Toxoplasma gondii infection. In the present study, we demonstrate that T. gondii-induced IGTP expression in vivo and IFN-gamma-driven synthesis of the protein in vitro are dependent on Stat1. Consistent with this observation, Stat1-deficient animals succumbed to T. gondii infection with the same rapid kinetics as IGTP(-/-) mice. To ascertain the cellular levels at which IGTP functions in host control of acute infection, we constructed reciprocal bone marrow chimeras between IGTP-deficient and wild-type mice. Resistance to infection was observed only when IGTP was present in both hematopoietic and nonhematopoietic compartments. To assess the possible contribution of IGTP to the maintenance of parasite latency, partial chemotherapy was used to allow the establishment of chronic infection in IGTP-deficient animals. Upon cessation of drug treatment, these animals showed delayed mortality compared with similarly infected and treated IFN-gamma-deficient or inducible nitric oxide synthase-deficient mice, which succumbed rapidly. Parallel experiments performed with drug-treated bone marrow chimeras supported a role for the hematopoietic compartment in this NO-dependent, IGTP-independent control of chronic infection. Taken together, our findings demonstrate that host resistance mediated by IGTP is a Stat1-induced function which in the case of T. gondii acts predominantly to restrict acute as opposed to chronic infection. This effector mechanism requires expression of IGTP in cells of both hematopoietic and nonhematopoietic origin. In contrast, in latent infection, hematopoietically derived cells mediate resistance by means of a largely NO-dependent pathway.

Structural determinants of the anti-HIV activity of a CCR5 antagonist derived from Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii possesses a protein, cyclophilin-18 (C-18), which binds to the chemokine receptor CCR5, induces interleukin-12 production from murine dendritic cells, and inhibits fusion and infectivity of human immunodeficiency virus 1 (HIV-1) R5 viruses by co-receptor antagonism. Site-directed mutagenesis was employed to identify the domains in C-18 responsible for its CCR5 binding and antiviral functions. To do so we focused on amino acid differences with Plasmodium falciparum cyclophilin, which, although 53% identical with C-18, has minimal binding activity for CCR5, and we generated 22 mutants with substitutions in the regions of non-homology located on the putative surface of the molecule. Two mutations situated on the face of C-18, predicted to be involved in its interaction with the ligand cyclosporin A, were shown to be critical for CCR5-binding and the inhibition of HIV-1 fusion and infectivity. In contrast, four mutations in C-18 specifically designed to abolish the peptidyl-prolyl cis-trans-isomerase activity of the protein failed to inactivate its CCR5 binding and HIV inhibitory activities. Interleukin-12 induction by C-18, on the other hand, was abrogated by mutations effecting either the CCR5 binding or enzymatic function of the molecule. These findings shed light on the structural basis of the molecular mimicry of the chemokine function by a pathogen-derived protein and provide a basis for further modification of C-18 into an antiviral agent.

Mice lacking myeloid differentiation factor 88 display profound defects in host resistance and immune responses to Mycobacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-deficient animals.

To assess the role of Toll-like receptor (TLR) signaling in host resistance to Mycobacterium avium infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88), as well as TLR2(-/-) and TLR4(-/-) animals, were infected with a virulent strain of M. avium, and bacterial burdens and immune responses were compared with those in wild-type (WT) animals. MyD88(-/-) mice failed to control acute and chronic M. avium growth and succumbed 9-14 wk postinfection. Infected TLR2(-/-) mice also showed increased susceptibility, but displayed longer survival and lower bacterial burdens than MyD88(-/-) animals, while TLR4(-/-) mice were indistinguishable from their WT counterparts. Histopathological examination of MyD88(-/-) mice revealed massive destruction of lung tissue not present in WT, TLR2(-/-), or TLR4(-/-) mice. In addition, MyD88(-/-) and TLR2(-/-), but not TLR4(-/-), mice displayed marked reductions in hepatic neutrophil infiltration during the first 2 h of infection. Although both MyD88(-/-) and TLR2(-/-) macrophages showed profound defects in IL-6, TNF, and IL-12p40 responses to M. avium stimulation in vitro, in vivo TNF and IL-12p40 mRNA induction was impaired only in infected MyD88(-/-) mice. Similarly, MyD88(-/-) mice displayed a profound defect in IFN-gamma response that was not evident in TLR2(-/-) or TLR4(-/-) mice or in animals deficient in IL-18. These findings indicate that resistance to mycobacterial infection is regulated by multiple MyD88-dependent signals in addition to those previously attributed to TLR2 or TLR4, and that these undefined elements play a major role in determining bacterial induced proinflammatory as well as IFN-gamma responses.

Induction of colitis by a CD4+ T cell clone specific for a bacterial epitope.

It is now well established that the intestinal flora plays an important role in the pathogenesis of inflammatory bowel disease (IBD). However, whether bacteria serve as the sole target of the immune response in this process or whether they act indirectly by triggering an anti-self response is still unclear. We have previously shown that specific pathogen-free IL-10-deficient (IL-10 KO) mice develop a T helper (Th1)-cytokine associated colitis after experimental infection with Helicobacter hepaticus. We here show that H. hepaticus Ag (SHelAg)-specific CD4+ Th1 clones transfer disease to H. hepaticus-infected T cell-deficient RAG KO hosts. Importantly, uninfected recipients of the SHelAg-specific clones did not develop intestinal inflammation, and a control Schistosoma mansoni-specific Th1 clone did not induce colitis upon transfer to infected RAG KO mice. The disease-inducing T cell clones recognized antigen(s) (Ag) specifically expressed by certain Helicobacter species as they responded when stimulated in vitro with H. hepaticus and Helicobacter typhlonius Ag, but not when cultured with Ag preparations from Helicobacter pylori, various non-helicobacter bacteria, or with cecal bacterial lysate from uninfected mice. Characterization of the Ag specificity of one of the clones showed that it reacts uniquely with a 15-mer peptide epitope on the flagellar hook protein (FlgE) of H. hepaticus presented by I-Ab. Together, our results demonstrate that colitis can be induced by clonal T cell populations that are highly specific for target Ag on intestinal bacteria, suggesting that an aberrant T cell response directed against gut flora is sufficient to trigger IBD.