Erythropoietin: a potent inducer of peripheral immuno/inflammatory modulation in autoimmune EAE

Background

Beneficial effects of short-term erythropoietin (EPO) therapy have been demonstrated in several animal models of acute neurologic injury, including experimental autoimmune encephalomyelitis (EAE)-the animal model of multiple sclerosis. We have found that EPO treatment substantially reduces the acute clinical paralysis seen in EAE mice and this improvement is accompanied by a large reduction in the mononuclear cell infiltration and downregulation of glial MHC class II expression within the inflamed CNS. Other reports have recently indicated that peripherally generated anti-inflammatory CD4⁺Foxp3⁺ regulatory T cells (Tregs) and the IL17-producing CD4+ T helper cell (Th17) subpopulations play key antagonistic roles in EAE pathogenesis. However, no information regarding the effects of EPO therapy on the behavior of the general mononuclear-lymphocyte population, Tregs or Th17 cells in EAE has emerged.

Methods and Findings

We first determined in vivo that EPO therapy markedly suppressed MOG specific T cell proliferation and sharply reduced the number of reactive dendritic cells (CD11c positive) in EAE lymph nodes during both inductive and later symptomatic phases of MOG_35–55 induced EAE. We then determined the effect in vivo of EPO on numbers of peripheral Treg cells and Th17 cells. We found that EPO treatment modulated immune balance in both the periphery and the inflamed spinal cord by promoting a large expansion in Treg cells, inhibiting Th17 polarization and abrogating proliferation of the antigen presenting dendritic cell population. Finally we utilized tissue culture assays to show that exposure to EPO in vitro similarly downregulated MOG-specific T cell proliferation and also greatly suppressed T cell production of pro-inflammatory cytokines.

Conclusions

Taken together, our findings reveal an important new locus whereby EPO induces substantial long-term tissue protection in the host through signaling to several critical subsets of immune cells that reside in the peripheral lymphatic system.     

A Novel Probiotic Mixture Exerts a Therapeutic Effect on Experimental Autoimmune Encephalomyelitis Mediated by IL-10 Producing Regulatory T Cells

Background

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

Methodology/Principal Findings

The preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-β1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4⁺CD25⁺ Tregs are involved in the suppressive effect induced by the lactobacilli mixture.

Conclusions/Significance

Our data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases.     

Effects of natalizumab treatment on Foxp3+ T regulatory cells

Background

Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients.

Methodology

A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.

Principal Findings

Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25^highCD127^lowFoxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.

Conclusions

We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.     

Rapamycin combined with anti-CD45RB mAb and IL-10 or with G-CSF induces tolerance in a stringent mouse model of islet transplantation

Background

A large pool of preexisting alloreactive effector T cells can cause allogeneic graft rejection following transplantation. However, it is possible to induce transplant tolerance by altering the balance between effector and regulatory T (Treg) cells. Among the various Treg-cell types, Foxp3⁺Treg and IL-10–producing T regulatory type 1 (Tr1) cells have frequently been associated with tolerance following transplantation in both mice and humans. Previously, we demonstrated that rapamycin+IL-10 promotes Tr1-cell–associated tolerance in Balb/c mice transplanted with C57BL/6 pancreatic islets. However, this same treatment was unsuccessful in C57BL/6 mice transplanted with Balb/c islets (classified as a stringent transplant model). We accordingly designed a protocol that would be effective in the latter transplant model by simultaneously depleting effector T cells and fostering production of Treg cells. We additionally developed and tested a clinically translatable protocol that used no depleting agent.

Methodology/Principal Findings

Diabetic C57BL/6 mice were transplanted with Balb/c pancreatic islets. Recipient mice transiently treated with anti-CD45RB mAb+rapamycin+IL-10 developed antigen-specific tolerance. During treatment, Foxp3⁺Treg cells were momentarily enriched in the blood, followed by accumulation in the graft and draining lymph node, whereas CD4⁺IL-10⁺IL-4⁻ T (i.e., Tr1) cells localized in the spleen. In long-term tolerant mice, only CD4⁺IL-10⁺IL-4⁻ T cells remained enriched in the spleen and IL-10 was key in the maintenance of tolerance. Alternatively, recipient mice were treated with two compounds routinely used in the clinic (namely, rapamycin and G-CSF); this drug combination promoted tolerance associated with CD4⁺IL-10⁺IL-4⁻ T cells.

Conclusions/Significance

The anti-CD45RB mAb+rapamycin+IL-10 combined protocol promotes a state of tolerance that is IL-10 dependent. Moreover, the combination of rapamycin+G-CSF induces tolerance and such treatment could be readily translatable into the clinic.     

Regulatory T cells in the pathogenesis and healing of chronic human dermal leishmaniasis caused by Leishmania (Viannia) species

Background

The inflammatory response is prominent in the pathogenesis of dermal leishmaniasis. We hypothesized that regulatory T cells (Tregs) may be diminished in chronic dermal leishmaniasis (CDL) and contribute to healing during treatment.

Methodology/Principal Findings

The frequency and functional capacity of Tregs were evaluated at diagnosis and following treatment of CDL patients having lesions of ≥6 months duration and asymptomatically infected residents of endemic foci. The frequency of CD4⁺CD25^hi cells expressing Foxp3 or GITR or lacking expression of CD127 in peripheral blood was determined by flow cytometry. The capacity of CD4⁺CD25⁺ cells to inhibit Leishmania-specific responses was determined by co-culture with effector CD4⁺CD25⁻ cells. The expression of FOXP3, IFNG, IL10 and IDO was determined in lesion and leishmanin skin test site biopsies by qRT-PCR. Although CDL patients presented higher frequency of CD4⁺CD25^hiFoxp3⁺ cells in peripheral blood and higher expression of FOXP3 at leishmanin skin test sites, their CD4⁺CD25⁺ cells were significantly less capable of suppressing antigen specific-IFN-γ secretion by effector cells compared with asymptomatically infected individuals. At the end of treatment, both the frequency of CD4⁺CD25^hiCD127⁻ cells and their capacity to inhibit proliferation and IFN-γ secretion increased and coincided with healing of cutaneous lesions. IDO was downregulated during healing of lesions and its expression was positively correlated with IFNG but not FOXP3.

Conclusions/Significance

The disparity between CD25^hiFoxp3⁺ CD4 T cell frequency in peripheral blood, Foxp3 expression at the site of cutaneous responses to leishmanin, and suppressive capacity provides evidence of impaired Treg function in the pathogenesis of CDL. Moreover, the concurrence of increased Leishmania-specific suppressive capacity with induction of a CD25^hiCD127⁻ subset of CD4 T cells during healing supports the participation of Tregs in the resolution of chronic dermal lesions. Treg subsets may therefore be relevant in designing immunotherapeutic strategies for recalcitrant dermal leishmaniasis caused by Leishmania (Viannia) species.     

Local induction of immunosuppressive CD8+ T cells in the gut-associated lymphoid tissues

Background

In contrast to intestinal CD4⁺ regulatory T cells (T_regs), the generation and function of immunomodulatory intestinal CD8⁺ T cells is less well defined. To dissect the immunologic mechanisms of CD8⁺ T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied.

Methodology and Principal Findings

HA-specific CD8⁺ T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3⁺ and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8⁺Foxp3⁺ T cells. Antigen-experienced CD8⁺ T cells in this transgenic mouse model suppressed the proliferation of CD8⁺ and CD4⁺ T cells in vitro. Gene expression analysis of suppressive HA-specific CD8⁺ T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4⁺ T_reg subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8⁺Foxp3⁺ T cells.

Conclusion and Significance

We demonstrate that gut specific antigen presentation is sufficient to induce CD8⁺ T_regs in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells.     

TGF-β induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction

Background

It has been reported that human FOXP3⁺ CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3⁺ Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs.

Methodology/Principal Findings

We generated anti-mouse LAP mAbs by immunizing TGF-β^−/− animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3⁺ CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4⁺CD25⁻ T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4⁺CD25⁻ T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3⁺ but also on T cells that remained Foxp3⁻ after TGF-β treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells.

Conclusions/Significance

Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-β. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.     

Regulatory T cell induction during Plasmodium chabaudi infection modifies the clinical course of experimental autoimmune encephalomyelitis

Background

Experimental autoimmune encephalomyelitis (EAE) is used as an animal model for human multiple sclerosis (MS), which is an inflammatory demyelinating autoimmune disease of the central nervous system characterized by activation of Th1 and/or Th17 cells. Human autoimmune diseases can be either exacerbated or suppressed by infectious agents. Recent studies have shown that regulatory T cells play a crucial role in the escape mechanism of Plasmodium spp. both in humans and in experimental models. These cells suppress the Th1 response against the parasite and prevent its elimination. Regulatory T cells have been largely associated with protection or amelioration in several autoimmune diseases, mainly by their capacity to suppress proinflammatory response.

Methodology/Principal Findings

In this study, we verified that CD4⁺CD25⁺ regulatory T cells (T regs) generated during malaria infection (6 days after EAE induction) interfere with the evolution of EAE. We observed a positive correlation between the reduction of EAE clinical symptoms and an increase of parasitemia levels. Suppression of the disease was also accompanied by a decrease in the expression of IL-17 and IFN-γ and increases in the expression of IL-10 and TGF-β1 relative to EAE control mice. The adoptive transfer of CD4⁺CD25⁺ cells from P. chabaudi-infected mice reduced the clinical evolution of EAE, confirming the role of these T regs.

Conclusions/Significance

These data corroborate previous findings showing that infections interfere with the prevalence and evolution of autoimmune diseases by inducing regulatory T cells, which regulate EAE in an apparently non-specific manner.     

Impaired thymic export and apoptosis contribute to regulatory T-cell defects in patients with chronic heart failure

Objective

Animal studies suggest that regulatory T (T_reg) cells play a beneficial role in ventricular remodeling and our previous data have demonstrated defects of T_reg cells in patients with chronic heart failure (CHF). However, the mechanisms behind T_reg-cell defects remained unknown. We here sought to elucidate the mechanism of T_reg-cell defects in CHF patients.

Methods and Results

We performed flow cytometry analysis and demonstrated reduced numbers of peripheral blood CD4⁺CD25⁺FOXP3⁺CD45RO⁻CD45RA⁺ naïve T_reg (nT_reg) cells and CD4⁺CD25⁺FOXP3⁺CD45RO⁺CD45RA⁻ memory T_reg (mT_reg) cells in CHF patients as compared with non-CHF controls. Moreover, the nT_reg/mT_reg ratio (p<0.01), CD4⁺CD25⁺FOXP3⁺CD45RO⁻ CD45RA⁺CD31⁺ recent thymic emigrant T_reg cell (RTE-T_reg) frequency (p<0.01), and T-cell receptor excision circle levels in T_reg cells (p<0.01) were lower in CHF patients than in non-CHF controls. Combined annexin-V and 7-AAD staining showed that peripheral T_reg cells from CHF patients exhibited increased spontaneous apoptosis and were more prone to interleukin (IL)-2 deprivation- and CD95 ligand-mediated apoptosis than those from non-CHF individuals. Furthermore, analyses by both flow cytometry and real-time polymerase chain reaction showed that T_reg-cell frequency in the mediastinal lymph nodes or Foxp3 expression in hearts of CHF patients was no higher than that of the non-CHF controls.

Conclusion

Our data suggested that the T_reg-cell defects of CHF patients were likely caused by decreased thymic output of nascent T_reg cells and increased susceptibility to apoptosis in the periphery.     

BAFF promotes Th17 cells and aggravates experimental autoimmune encephalomyelitis

Background

BAFF, in addition to promoting B cell survival and differentiation, may affect T cells. The objective of this study was to determine the effect of BAFF on Th17 cell generation and its ramifications for the Th17 cell-driven disease, EAE.

Methodology/Principal Findings

Th17 cells were increased in BAFF-Tg B6 (B6.BTg) mice and decreased in B6.Baff^−/− mice. Th17 cells in B6.Baff^−/− mice bearing a BAFF Tg (B6.Baff^−/−.BTg mice) were identical to those in B6.BTg mice, indicating that membrane BAFF is dispensable for Th17 cell generation as long as soluble BAFF is plentiful. In T + non-T cell criss-cross co-cultures, Th17 cell generation was greatest in cultures containing B6.BTg T cells and lowest in cultures containing B6.Baff^−/− T cells, regardless of the source of non-T cells. In cultures containing only T cells, Th17 cell generation followed an identical pattern. CD4⁺ cell expression of CD126 (IL-6R α chain) was increased in B6.BTg mice and decreased in B6.Baff^−/− mice, and activation of STAT3 following stimulation with IL-6 + TGF-β was also greatest in B6.BTg cells and lowest in B6.Baff^−/− cells. EAE was clinically and pathologically most severe in B6.BTg mice and least severe in B6.Baff^−/− mice and correlated with MOG_35–55 peptide-induced Th17 cell responses.

Conclusions/Significance

Collectively, these findings document a contribution of BAFF to pathogenic Th17 cell responses and suggest that BAFF antagonism may be efficacious in Th17 cell-driven diseases.     

Lymphocyte subpopulations in lymph nodes and peripheral blood: a comparison between patients with stable angina and acute coronary syndrome

Objective

Atherosclerosis is characterized by a chronic inflammatory response involving activated T cells and impairment of natural killer (NK) cells. An increased T cell activity has been associated with plaque instability and risk of acute cardiac events. Lymphocyte analyses in blood are widely used to evaluate the immune status. However, peripheral blood contains only a minor proportion of lymphocytes. In this study, we hypothesized that thoracic lymph nodes from patients with stable angina (SA) and acute coronary syndrome (ACS) might add information to peripheral blood analyses.

Methods

Peripheral blood and lymph nodes were collected during coronary by-pass surgery in 13 patients with SA and 13 patients with ACS. Lymphocyte subpopulations were assessed by flow cytometry using antibodies against CD3, CD4, CD8, CD19, CD16/56, CD25, Foxp3, CD69, HLA-DR, IL-18 receptor (R) and CCR4.

Results

Lymph nodes revealed a lymphocyte subpopulation profile substantially differing from that in blood including a higher proportion of B cells, lower proportions of CD8⁺ T cells and NK cells and a 2-fold higher CD4/CD8 ratio. CD4⁺CD69⁺ cells as well as Foxp3⁺ regulatory T cells were markedly enriched in lymph nodes (p<0.001) while T helper 1-like (CD4⁺IL-18R+) cells were more frequent in blood (p<0.001). The only significant differences between ACS and SA patients involved NK cells that were reduced in the ACS group. However, despite being reduced, the NK cell fraction in ACS patients contained a significantly higher proportion of IL-18R⁺ cells compared with SA patients (p<0.05).

Conclusion

There were several differences in lymphocyte subpopulations between blood and lymph nodes. However, the lymphocyte perturbations in peripheral blood of ACS patients compared with SA patients were not mirrored in lymph nodes. The findings indicate that lymph node analyses in multivessel coronary artery disease may not reveal any major changes in the immune response that are not detectable in blood.     

Assessing the role of CD103 in immunity to an intestinal helminth parasite

Background

In the intestine, the integrin CD103 is expressed on a subset of T regulatory (T_reg) cells and a population of dendritic cells (DCs) that produce retinoic acid and promote immune homeostasis. However, the role of CD103 during intestinal helminth infection has not been tested.

Methodology/Principal Findings

We demonstrate that CD103 is dispensable for the development of protective immunity to the helminth parasite Trichuris muris. While we observed an increase in the frequency of CD103⁺ DCs in the lamina propria (LP) following acute high-dose infection with Trichuris, lack of CD103 had no effect on the frequency of CD11c⁺ DCs in the LP or mesenteric lymph nodes (mLN). CD103-deficient (CD103^−/−) mice develop a slightly increased and earlier T cell response but resolve infection with similar kinetics to control mice. Similarly, low-dose chronic infection of CD103^−/− mice with Trichuris resulted in no significant difference in immunity or parasite burden. Absence of CD103 also had no effect on the frequency of CD4⁺CD25⁺Foxp3⁺ T_reg cells in the mLN or LP.

Conclusions/Significance

These results suggest that CD103 is dispensable for intestinal immunity during helminth infection. Furthermore, lack of CD103 had no effect on DC or T_reg recruitment or retention within the large intestine.     

Cyp26b1 regulates retinoic acid-dependent signals in T cells and its expression is inhibited by transforming growth factor-β

Background

The vitamin A metabolite, retinoic acid (RA), plays important roles in the regulation of lymphocyte properties. Dendritic cells in gut-related lymphoid organs can produce RA, thereby imprinting gut-homing specificity on T cells and enhancing transforming growth factor (TGF)-β-dependent induction of Foxp3⁺ regulatory T cells upon antigen presentation. In general, RA concentrations in cells and tissues are regulated by its degradation as well. However, it remained unclear if T cells could actively catabolize RA.

Methodology/Principal Findings

We assessed the expression of known RA-catabolizing enzymes in T cells from mouse lymphoid tissues. Antigen-experienced CD44⁺ T cells in gut-related lymphoid organs selectively expressed Cyp26b1, a member of the cytochrome P450 family 26. However, T cells in the spleen or skin-draining lymph nodes did not significantly express Cyp26b1. Accordingly, physiological levels of RA (1–10 nM) could induce Cyp26b1 expression in naïve T cells upon activation in vitro, but could not do so in the presence of TGF-β. Overexpression of Cyp26b1 significantly suppressed the RA effect to induce expression of the gut-homing receptor CCR9 on T cells. On the other hand, knocking down Cyp26b1 gene expression with small interfering RNA or inhibiting CYP26 enzymatic activity led to enhancement of the RA-induced CCR9 expression.

Conclusions/Significance

Our data demonstrate a role for CYP26B1 in regulating RA-dependent signals in activated T cells but not during TGF-β-dependent differentiation to Foxp3⁺ regulatory T cells. Aberrant expression of CYP26B1 may disturb T cell trafficking and differentiation in the gut and its related lymphoid organs.     

Interleukin-10 (IL-10) pathway: genetic variants and outcomes of HIV-1 infection in African American adolescents

Background

Immunological and clinical outcomes can vary considerably at the individual and population levels during both treated and untreated HIV-1 infection. Cytokines encoded by the interleukin-10 gene (IL10) family have broad immunomodulatory function in viral persistence, and several SNPs in the IL10 promoter sequence have been reported to influence pathogenesis or acquisition of HIV-1 infection.

Methodology/Principal Findings

We examined 104 informative SNPs in IL10, IL19, IL20, IL24, IL10RA and IL10RB among 250 HIV-1 seropositive and 106 high-risk seronegative African American adolescents in the REACH cohort. In subsequent evaluation of five different immunological and virological outcomes related to HIV-1 infection, 25 SNPs were associated with a single outcome and three were associated with two different outcomes. One SNP, rs2243191 in the IL19 open reading frame (Ser to Phe substitution) was associated with CD4⁺ T-cell increase during treatment. Another SNP rs2244305 in IL10RB (in strong linkage disequilibrium with rs443498) was associated with an initial decrease in CD4⁺ T-cell by 23±9% and 29±9% every 3 months (for AA and AG genotypes, respectively, compared with GG) during ART-free period. These associations were reversed during treatment, as CD4⁺ T-cell increased by 31±0.9% and 17±8% every 3 months for AA and AG genotype, respectively.

Conclusions/Significance

In African Americans, variants in IL10 and related genes might influence multiple outcomes of HIV-1 infection, especially immunological response to HAART. Fine mapping coupled with analysis of gene expression and function should help reveal the immunological importance of the IL10 gene family to HIV-1/AIDS.     

Early interferon-γ production in human lymphocyte subsets in response to nontyphoidal Salmonella demonstrates inherent capacity in innate cells

Background

Nontyphoidal Salmonellae frequently cause life-threatening bacteremia in sub-Saharan Africa. Young children and HIV-infected adults are particularly susceptible. High case-fatality rates and increasing antibiotic resistance require new approaches to the management of this disease. Impaired cellular immunity caused by defects in the T helper 1 pathway lead to intracellular disease with Salmonella that can be countered by IFNγ administration. This report identifies the lymphocyte subsets that produce IFNγ early in Salmonella infection.

Methodology

Intracellular cytokine staining was used to identify IFNγ production in blood lymphocyte subsets of ten healthy adults with antibodies to Salmonella (as evidence of immunity to Salmonella), in response to stimulation with live and heat-killed preparations of the {"type":"entrez-nucleotide","attrs":{"text":"D23580","term_id":"427513","term_text":"D23580"}}D23580 invasive African isolate of Salmonella Typhimurium. The absolute number of IFNγ-producing cells in innate, innate-like and adaptive lymphocyte subpopulations was determined.

Principal Findings

Early IFNγ production was found in the innate/innate-like lymphocyte subsets: γδ-T cells, NK cells and NK-like T cells. Significantly higher percentages of such cells produced IFNγ compared to adaptive αβ-T cells (Student''s t test, P<0.001 and ≤0.02 for each innate subset compared, respectively, with CD4⁺- and CD8⁺-T cells). The absolute numbers of IFNγ-producing cells showed similar differences. The proportion of IFNγ-producing γδ-T cells, but not other lymphocytes, was significantly higher when stimulated with live compared with heat-killed bacteria (P<0.0001).

Conclusion/Significance

Our findings indicate an inherent capacity of innate/innate-like lymphocyte subsets to produce IFNγ early in the response to Salmonella infection. This may serve to control intracellular infection and reduce the threat of extracellular spread of disease with bacteremia which becomes life-threatening in the absence of protective antibody. These innate cells may also help mitigate against the effect on IFNγ production of depletion of Salmonella-specific CD4⁺-T lymphocytes in HIV infection.     

Caspase-3 is transiently activated without cell death during early antigen driven expansion of CD8(+) T cells in vivo

Background

CD8⁺ T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8⁺ T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8⁺ T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8⁺ T cell responses has yet to be examined.

Methods and Findings

We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8⁺ T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8⁺ T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3^hi and caspase-3^low CD8⁺ T cells. The expression of active caspase-3 peaked before effector phenotype (CD62L^low) CD8⁺ T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8⁺ cells remained active caspase-3^low throughout the contraction phase.

Conclusions

Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8⁺ T cells. Furthermore, the contraction of CD8⁺ T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3.     

Visceral adipose inflammation in obesity is associated with critical alterations in tregulatory cell numbers

Background

The development of insulin resistance (IR) in mouse models of obesity and type 2 diabetes mellitus (DM) is characterized by progressive accumulation of inflammatory macrophages and subpopulations of T cells in the visceral adipose. Regulatory T cells (Tregs) may play a critical role in modulating tissue inflammation via their interactions with both adaptive and innate immune mechanisms. We hypothesized that an imbalance in Tregs is a critical determinant of adipose inflammation and investigated the role of Tregs in IR/obesity through coordinated studies in mice and humans.

Methods and Findings

Foxp3-green fluorescent protein (GFP) “knock-in” mice were randomized to a high-fat diet intervention for a duration of 12 weeks to induce DIO/IR. Morbidly obese humans without overt type 2 DM (n = 13) and lean controls (n = 7) were recruited prospectively for assessment of visceral adipose inflammation. DIO resulted in increased CD3⁺CD4⁺, and CD3⁺CD8⁺ cells in visceral adipose with a striking decrease in visceral adipose Tregs. Treg numbers in visceral adipose inversely correlated with CD11b⁺CD11c⁺ adipose tissue macrophages (ATMs). Splenic Treg numbers were increased with up-regulation of homing receptors CXCR3 and CCR7 and marker of activation CD44. In-vitro differentiation assays showed an inhibition of Treg differentiation in response to conditioned media from inflammatory macrophages. Human visceral adipose in morbid obesity was characterized by an increase in CD11c⁺ ATMs and a decrease in foxp3 expression.

Conclusions

Our experiments indicate that obesity in mice and humans results in adipose Treg depletion. These changes appear to occur via reduced local differentiation rather than impaired homing. Our findings implicate a role for Tregs as determinants of adipose inflammation.