The cytokines interleukin 27 and interferon-γ promote distinct Treg cell populations required to limit infection-induced pathology.

Interferon-γ (IFN-γ) promotes a population of T-bet(+) CXCR3(+) regulatory T (Treg) cells that limit T helper 1 (Th1) cell-mediated pathology. Our studies demonstrate that interleukin-27 (IL-27) also promoted expression of T-bet and CXCR3 in Treg cells. During infection with Toxoplasma gondii, a similar population emerged that limited T cell responses and was dependent on IFN-γ in the periphery but on IL-27 at mucosal sites. Transfer of Treg cells ameliorated the infection-induced pathology observed in Il27(-/-) mice, and this was dependent on their ability to produce IL-10. Microarray analysis revealed that Treg cells exposed to either IFN-γ or IL-27 have distinct transcriptional profiles. Thus, IFN-γ and IL-27 have different roles in Treg cell biology and IL-27 is a key cytokine that promotes the development of Treg cells specialized to control Th1 cell-mediated immunity at local sites of inflammation.

CD11c-Expressing Cells Affect Regulatory T Cell Behavior in the Meninges during Central Nervous System Infection.

Regulatory T cells (Tregs) play an important role in the CNS during multiple infections, as well as autoimmune inflammation, but the behavior of this cell type in the CNS has not been explored. In mice, infection with Toxoplasma gondii leads to a Th1-polarized parasite-specific effector T cell response in the brain. Similarly, Tregs in the CNS during T. gondii infection are Th1 polarized, as exemplified by their T-bet, CXCR3, and IFN-γ expression. Unlike effector CD4+ T cells, an MHC class II tetramer reagent specific for T. gondii did not recognize Tregs isolated from the CNS. Likewise, TCR sequencing revealed minimal overlap in TCR sequence between effector T cells and Tregs in the CNS. Whereas effector T cells are found in the brain parenchyma where parasites are present, Tregs were restricted to the meninges and perivascular spaces. The use of intravital imaging revealed that activated CD4+ T cells within the meninges were highly migratory, whereas Tregs moved more slowly and were found in close association with CD11c+ cells. To test whether the behavior of Tregs in the meninges is influenced by interactions with CD11c+ cells, mice were treated with anti-LFA-1 Abs to reduce the number of CD11c+ cells in this space. The anti-LFA-1 treatment led to fewer contacts between Tregs and the remaining CD11c+ cells and increased the speed of Treg migration. These data suggest that Tregs are anatomically restricted within the CNS, and their interaction with CD11c+ populations regulates their local behavior during T. gondii infection.

Behavior of parasite-specific effector CD8+ T cells in the brain and visualization of a kinesis-associated system of reticular fibers.

To understand lymphocyte behavior in the brain, we used two-photon microscopy to visualize effector CD8(+) T cells during toxoplasmic encephalitis. These cells displayed multiple behaviors with two distinct populations of cells apparent: one with a constrained pattern of migration and one with a highly migratory subset. The proportion of these populations varied over time associated with changes in antigen availability as well as T cell expression of the inhibitory receptor PD1. Unexpectedly, the movement of infiltrating cells was closely associated with an infection-induced reticular system of fibers. This observation suggests that, whereas in other tissues pre-existing scaffolds exist that guide lymphocyte migration, in the brain specialized structures are induced by inflammation that guide migration of T cells in this immune-privileged environment.

Generalized Lévy walks and the role of chemokines in migration of effector CD8+ T cells.

Chemokines have a central role in regulating processes essential to the immune function of T cells, such as their migration within lymphoid tissues and targeting of pathogens in sites of inflammation. Here we track T cells using multi-photon microscopy to demonstrate that the chemokine CXCL10 enhances the ability of CD8+ T cells to control the pathogen Toxoplasma gondii in the brains of chronically infected mice. This chemokine boosts T-cell function in two different ways: it maintains the effector T-cell population in the brain and speeds up the average migration speed without changing the nature of the walk statistics. Notably, these statistics are not Brownian; rather, CD8+ T-cell motility in the brain is well described by a generalized Lévy walk. According to our model, this unexpected feature enables T cells to find rare targets with more than an order of magnitude more efficiency than Brownian random walkers. Thus, CD8+ T-cell behaviour is similar to Lévy strategies reported in organisms ranging from mussels to marine predators and monkeys, and CXCL10 aids T cells in shortening the average time taken to find rare targets.

Diverse roles for T-bet in the effector responses required for resistance to infection.

The transcription factor T-bet has been most prominently linked to NK and T cell production of IFN-γ, a cytokine required for the control of a diverse array of intracellular pathogens. Indeed, in mice challenged with the parasite Toxoplasma gondii, NK and T cell responses are characterized by marked increases of T-bet expression. Unexpectedly, T-bet(-/-) mice infected with T. gondii develop a strong NK cell IFN-γ response that controls parasite replication at the challenge site, but display high parasite burdens at secondary sites colonized by T. gondii and succumb to infection. The loss of T-bet had a modest effect on T cell production of IFN-γ but did not impact on the generation of parasite-specific T cells. However, the absence of T-bet resulted in lower T cell expression of CD11a, Ly6C, KLRG-1, and CXCR3 and fewer parasite-specific T cells at secondary sites of infection, associated with a defect in parasite control at these sites. Together, these data highlight T-bet-independent pathways to IFN-γ production and reveal a novel role for this transcription factor in coordinating the T cell responses necessary to control this infection in peripheral tissues.

The aryl hydrocarbon receptor promotes IL-10 production by NK cells.

The cytokine IL-10 has an important role in limiting inflammation in many settings, including toxoplasmosis. In the present studies, an IL-10 reporter mouse was used to identify the sources of this cytokine following challenge with Toxoplasma gondii. During infection, multiple cell types expressed the IL-10 reporter but NK cells were a major early source of this cytokine. These IL-10 reporter(+) NK cells expressed high levels of the IL-12 target genes T-bet, KLRG1, and IFN-γ, and IL-12 depletion abrogated reporter expression. However, IL-12 signaling alone was not sufficient to promote NK cell IL-10, and activation of the aryl hydrocarbon receptor (AHR) was also required for maximal IL-10 production. NK cells basally expressed the AHR, relevant chaperone proteins, and the AHR nuclear translocator, which heterodimerizes with the AHR to form a competent transcription factor. In vitro studies revealed that IL-12 stimulation increased NK cell AHR levels, and the AHR and AHR nuclear translocator were required for optimal production of IL-10. Additionally, NK cells isolated from T. gondii-infected Ahr(-/-) mice had impaired expression of IL-10, which was associated with increased resistance to this infection. Taken together, these data identify the AHR as a critical cofactor involved in NK cell production of IL-10.

Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella.

BACKGROUND: Autotransporter proteins represent a treasure trove for molecular engineers who modify Gram-negative bacteria for the export or secretion of foreign proteins across two membrane barriers. A particularly promising direction is the development of autotransporters as antigen display or secretion systems. Immunologists have been using ovalbumin as a reporter antigen for years and have developed sophisticated tools to detect specific T cells that respond to ovalbumin. Although ovalbumin-expressing bacteria are being used to trace T cell responses to colonizing or invading pathogens, current constructs for ovalbumin presentation have not been optimized. RESULTS: The activation of T helper cells in response to ovalbumin was improved by displaying the OVA-CD4 reporter epitope as a multimer on the surface of Salmonella and fused to the autotransporter MisL. Expression was optimized by including tandem in vivo promoters and two post-segregational killing systems for plasmid stabilization. CONCLUSIONS: The use of an autotransporter protein to present relevant epitope repeats on the surface of bacteria, combined with additional techniques favoring stable and efficient in vivo transcription, optimizes antigen presentation to T cells. The technique of multimeric epitope surface display should also benefit the development of new Salmonella or other enterobacterial vaccines.

Replication and distribution of Toxoplasma gondii in the small intestine after oral infection with tissue cysts.

Natural infection by Toxoplasma gondii occurs via oral ingestion of tissue cysts that rupture in the small intestine, releasing zoites that infect locally before disseminating throughout the host. The studies presented here used fluorescent parasites combined with flow cytometry and multiphoton microscopy techniques to understand the events associated with parasite replication in the mucosa. At 3 days postinfection with tissue cysts, parasites were localized in small foci and flow cytometry revealed parasites present in macrophages, neutrophils, and monocytes in the lamina propria. By day 6 postinfection, there were large foci of replicating parasites; however, foci unexpectedly varied in the number of villi involved and were associated with the presence of viable tachyzoites within the intestinal lumen. Consistent with the flow cytometry data, neutrophils and monocytes in the lamina propria were preferentially associated with parasite plaques. In contrast, dendritic cells comprised a small fraction of the infected immune cell population and were localized at the periphery of parasite plaques. Together, these findings reveal the formation of localized sites of parasite replication and inflammation early during infection and suggest that sustained replication of T. gondii in the gut may be a function of pathogen luminal spread.

Analysis of behavior and trafficking of dendritic cells within the brain during toxoplasmic encephalitis.

Under normal conditions the immune system has limited access to the brain; however, during toxoplasmic encephalitis (TE), large numbers of T cells and APCs accumulate within this site. A combination of real time imaging, transgenic reporter mice, and recombinant parasites allowed a comprehensive analysis of CD11c+ cells during TE. These studies reveal that the CNS CD11c+ cells consist of a mixture of microglia and dendritic cells (DCs) with distinct behavior associated with their ability to interact with parasites or effector T cells. The CNS DCs upregulated several chemokine receptors during TE, but none of these individual receptors tested was required for migration of DCs into the brain. However, this process was pertussis toxin sensitive and dependent on the integrin LFA-1, suggesting that the synergistic effect of signaling through multiple chemokine receptors, possibly leading to changes in the affinity of LFA-1, is involved in the recruitment/retention of DCs to the CNS and thus provides new insights into how the immune system accesses this unique site.

Dendritic cells distinguish individual chemokine signals through CCR7 and CXCR4.

Dendritic cells (DCs) respond to chemotactic signals to migrate from sites of infection to secondary lymphoid organs where they initiate the adaptive immune response. The key chemokines directing their migration are CCL19, CCL21, and CXCL12, but how signals from these chemokines are integrated by migrating cells is poorly understood. Using a microfluidic device, we presented single and competing chemokine gradients to murine bone-marrow derived DCs in a controlled, time-invariant microenvironment. Experiments performed with counter-gradients revealed that CCL19 is 10-100-fold more potent than CCL21 or CXCL12. Interestingly, when the chemoattractive potencies of opposing gradients are matched, cells home to a central region in which the signals from multiple chemokines are balanced; in this region, cells are motile but display no net displacement. Actin and myosin inhibitors affected the speed of crawling but not directed motion, whereas pertussis toxin inhibited directed motion but not speed. These results provide fundamental insight into the processes that DCs use to migrate toward and position themselves within secondary lymphoid organs.

Dynamic Imaging of CD8(+) T cells and dendritic cells during infection with Toxoplasma gondii.

To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.

Self-written waveguide in methylene blue sensitized poly(vinyl alcohol)/acrylamide photopolymer material.

We report the observation of a self-written waveguide inside a bulk methylene blue sensitized poly/vinyl alcohol)/acrylamide photopolymer material. Light from a low power He-Ne laser is focused into the material, and the evolution of the beam is monitored. The refractive index of the material is modulated in the region of high intensity due to photobleaching and photopolymerization effects occurring in the material. As a result, the beam propagates through the medium without any diffraction effects.

TLR4-Dependent Secretion by Hepatic Stellate Cells of the Neutrophil-Chemoattractant CXCL1 Mediates Liver Response to Gut Microbiota.

BACKGROUND & AIMS: The gut microbiota significantly influences hepatic immunity. Little is known on the precise mechanism by which liver cells mediate recognition of gut microbes at steady state. Here we tested the hypothesis that a specific liver cell population was the sensor and we aimed at deciphering the mechanism by which the activation of TLR4 pathway would mediate liver response to gut microbiota. METHODS: Using microarrays, we compared total liver gene expression in WT versus TLR4 deficient mice. We performed in situ localization of the major candidate protein, CXCL1. With an innovative technique based on cell sorting, we harvested enriched fractions of KCs, LSECs and HSCs from the same liver. The cytokine secretion profile was quantified in response to low levels of LPS (1ng/mL). Chemotactic activity of stellate cell-derived CXCL1 was assayed in vitro on neutrophils upon TLR4 activation. RESULTS: TLR4 deficient liver had reduced levels of one unique chemokine, CXCL1 and subsequent decreased of neutrophil counts. Depletion of gut microbiota mimicked TLR4 deficient phenotype, i.e., decreased neutrophils counts in the liver. All liver cells were responsive to low levels of LPS, but hepatic stellate cells were the major source of chemotactic levels of CXCL1. Neutrophil migration towards secretory hepatic stellate cells required the TLR4 dependent secretion of CXCL1. CONCLUSIONS: Showing the specific activation of TLR4 and the secretion of one major functional chemokine-CXCL1, the homolog of human IL-8-, we elucidate a new mechanism in which Hepatic Stellate Cells play a central role in the recognition of gut microbes by the liver at steady state.

STAT1 Signaling in Astrocytes Is Essential for Control of Infection in the Central Nervous System.

The local production of gamma interferon (IFN-γ) is important to control Toxoplasma gondii in the brain, but the basis for these protective effects is not fully understood. The studies presented here reveal that the ability of IFN-γ to inhibit parasite replication in astrocytes in vitro is dependent on signal transducer and activator of transcription 1 (STAT1) and that mice that specifically lack STAT1 in astrocytes are unable to limit parasite replication in the central nervous system (CNS). This susceptibility is associated with a loss of antimicrobial pathways and increased cyst formation in astrocytes. These results identify a critical role for astrocytes in limiting the replication of an important opportunistic pathogen. IMPORTANCE: Astrocytes are the most numerous cell type in the brain, and they are activated in response to many types of neuroinflammation, but their function in the control of CNS-specific infection is unclear. The parasite Toxoplasma gondii is one of the few clinically relevant microorganisms that naturally infects astrocytes, and the studies presented here establish that the ability of astrocytes to inhibit parasite replication is essential for the local control of this opportunistic pathogen. Together, these studies establish a key role for astrocytes as effector cells and in the coordination of many aspects of the protective immune response that operates in the brain.

IFN-gamma bioassay: development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice.

Interferon-gamma (IFN-gamma) is an important immunomodulatory and pleiotropic cytokine produced, primarily, by activated T lymphocytes and natural killer (NK) cells. We have devised a nitric oxide (NO)-based bioassay for mouse IFN-gamma using resident peritoneal exudate cells (PECs) from C57BL/6 mice. Comparison with three existing bioassays demonstrated that this assay was very sensitive and detected IFN-gamma in the linear range of approximately 0.03-0.25 U/ml. Other cytokines, e.g. interleukin (IL)-2, IL-4, IL-6, IFN-alpha/beta and tumor necrosis factor-alpha (TNF-alpha), either alone or in combination with IFN-gamma, did not greatly modulate NO levels produced by resident peritoneal exudate cells. The presence of exogenous NO(3)(-) and H(2)O(2) did not interfere with the IFN-gamma induced NO production and detection. We also showed that the effect of lipopolysaccharide (LPS), which may be present in samples, could be suppressed by the use of Polymyxin B in the bioassay. The high sensitivity of the bioassay permitted the detection of low amounts of IFN-gamma in 1% mouse serum. In addition, this assay reproducibly detected bioactive IFN-gamma amounts in supernatants of activated T cells. The increase in IFN-gamma production by activated T cells in response to CD28 costimulation was approximately 3-fold by this bioassay and approximately 5-fold by ELISA. In summary, we have devised a simple, sensitive, inexpensive and high throughput method for the reproducible detection of bioactive IFN-gamma.

Advances in imaging the innate and adaptive immune response to Toxoplasma gondii.

Toxoplasma gondii is an intracellular protozoan parasite that infects a wide variety of warm-blooded hosts and can have devastating effects in the developing fetus as well as the immunocompromised host. An appreciation of how this organism interacts with the host immune system is crucial to understanding the pathogenesis of this disease. The last decade has been marked by the application of various imaging techniques, such as bioluminescent imaging as well as confocal and multiphoton microscopy to study toxoplasmosis. The ability to manipulate parasites to express fluorescent/bioluminescent markers or model antigens/enzymes combined with the development of reporter mice that allow the detection of distinct immune populations have been crucial to the success of many of these studies. These approaches have permitted the visualization of parasites and immune cells in real-time and provided new insights into the nature of host-pathogen interactions. This article highlights some of the advances in imaging techniques, their strengths and weaknesses, and how these techniques have impacted our understanding of the interaction between parasites and various immune populations during toxoplasmosis.

Immune role of hepatic TLR-4 revealed by orthotopic mouse liver transplantation.

UNLABELLED: Activated CD8+ T cells migrate to the liver at the end of an immune response and go through apoptosis there, but this mechanism is impaired in mice lacking Toll-like receptor-4. This allowed us to test the importance of liver trapping in an ongoing immune response. In the absence of Toll-like receptor-4, reduced liver accumulation was associated with an increase in the circulating CD8+ T cell pool, more long-lived memory T cells and increased CD8+ T cell memory responses. Using experimental orthotopic liver transplantation, we showed that the effect of Toll-like receptor-4 on the formation of the CD8+ T cell memory resides in the liver. CONCLUSION: These studies reveal a new function for the liver, which is to regulate the magnitude of T cell memory responses through a Toll-like receptor-4-dependent mechanism.

Enhancement of diffraction efficiency and storage life of poly(vinyl chloride)-based optical recording medium with incorporation of an electron donor.

The diffraction efficiency, sensitivity, and storage life of methylene blue-sensitized poly(vinyl chloride) film was improved by the addition of an electron donor in the matrix. The addition of pyridine enhanced the diffraction efficiency by two times, and storage life of the gratings was increased to 2-3 days.

Kupffer cell heterogeneity: functional properties of bone marrow derived and sessile hepatic macrophages.

Kupffer cells form a large intravascular macrophage bed in the liver sinusoids. The differentiation history and diversity of Kupffer cells is disputed; some studies argue that they are derived from blood monocytes, whereas others support a local origin from intrahepatic precursor cells. In the present study, we used both flow cytometry and immunohistochemistry to distinguish 2 subsets of Kupffer cells that were revealed in the context both of bone marrow transplantation and of orthotopic liver transplantation. One subset was radiosensitive and rapidly replaced from hematogenous precursors, whereas the other was relatively radioresistant and long-lived. Both were phagocytic but only the former population was recruited into inflammatory foci in response to CD8(+) T-cell activation. We propose the name "sessile" for the radioresistant Kupffer cells that do not participate in immunoinflammatory reactions. However, we found no evidence that these sessile Kupffer cells arise from immature intrahepatic precursors. Our conclusions resolve a long-standing controversy and explain how different experimental approaches may reveal one or both of these subsets.

Cellular and molecular mechanisms of liver tolerance.

The liver exhibits a distinctive form of immune privilege, termed liver tolerance, in which orthotopic liver transplantation results in systemic donor-specific T-cell tolerance, while antigens introduced either into hepatocytes or via the portal vein also cause tolerance. Here we argue that the fundamental mechanism driving liver tolerance is likely to be the continuous exposure of diverse liver cell types to endotoxin, derived from the intestinal bacteria. This exposure promotes the expression of a set of cytokines, antigen-presenting molecules, and costimulatory signals that impose T-cell inactivation, partly via effects on liver antigen-presenting cells. The evidence favors clonal deletion mechanisms and is consistent with a role for regulatory T cells but does not support either anergy or immune deviation as important factors in liver tolerance.