TLR2 Is a Negative Regulator of Th17 Cells and Tissue Pathology in a Pulmonary Model of Fungal Infection

To study the role of TLR2 in a experimental model of chronic pulmonary infection, TLR2-deficient and wild-type mice were intratracheally infected with Paracoccidioides brasiliensis, a primary fungal pathogen. Compared with control, TLR2(-/-) mice developed a less severe pulmonary infection and decreased NO synthesis. Equivalent results were detected with in vitro-infected macrophages. Unexpectedly, despite the differences in fungal loads both mouse strains showed equivalent survival times and severe pulmonary inflammatory reactions. Studies on lung-infiltrating leukocytes of TLR2(-/-) mice demonstrated an increased presence of polymorphonuclear neutrophils that control fungal loads but were associated with diminished numbers of activated CD4(+) and CD8(+) T lymphocytes. TLR2 deficiency leads to minor differences in the levels of pulmonary type 1 and type 2 cytokines, but results in increased production of KC, a CXC chemokine involved in neutrophils chemotaxis, as well as TGF-beta, IL-6, IL-23, and IL-17 skewing T cell immunity to a Th17 pattern. In addition, the preferential Th17 immunity of TLR2(-/-) mice was associated with impaired expansion of regulatory CD4(+)CD25(+)FoxP3(+) T cells. This is the first study to show that TLR2 activation controls innate and adaptive immunity to P. brasiliensis infection. TLR2 deficiency results in increased Th17 immunity associated with diminished expansion of regulatory T cells and increased lung pathology due to unrestrained inflammatory reactions. The Journal of Immunology, 2009, 183: 1279-1290.

The Impact of Vitamin A Supplementation on the Immune System of Vitamin A-deficient Children

Background & Aims: To investigate the effect of vitamin A supplementation on parameters of the immune system of vitamin A-deficient children. Methods: The study was carried out in four phases: 1) determination of serum retinol in 631 children from 36 to 83 months of age; 2) assessment of immunological markers [immunoglobulins and complement fractions, immunophenotyping of T and B lymphocytes, and natural killer (NK) cells], blood count, and serum ferritin of 52 vitamin A-deficient children (serum retinol <0.70 mu mol/L); 3) supplementation of the 52 deficient children with 200,000 IU of vitamin A; 4) determination of serum retinol and the immunological parameters 2 months after vitamin A supplementation. Results: Before vitamin A supplementation, 24.0% of the children were anemic and 4.3 %had reduced ferritin concentrations. There was no significant difference between mean values of retinol according to the presence/absence of anemia. The mean values of the humoral and cellular immunological parameters did not show a statistically significant difference before and after supplementation with vitamin A. Children with concomitant hypovitaminosis A and anemia presented a significant increase in absolute CD4 and CD8 T-cell counts after vitamin A supplementation (p < 0.05). Conclusion: Vitamin A had an effect on the recruitment of T and B lymphocytes to the circulation of children with hypovitaminosis A and anemia.

Glucose metabolism by lymphocytes, macrophages, and tumor cells from Walker 256 tumor-bearing rats supplemented with fish oil for one generation

Here we investigated the effect of lifelong supplementation of the diet with coconut fat (CO, rich in saturated fatty acids) or fish oil (170, rich in n-3 polyunsaturated fatty acids) on tumor growth and lactate production from glucose in Walker 256 tumor cells, peritoneal macrophages, spleen, and gut-associated lymphocytes. Female Wistar rats were supplemented with CO or FO prior to mating and then throughout pregnancy and gestation and then the male offspring were supplemented from weaning until 90 days of age. Then they were inoculated subcutaneously with Walker 256 tumor cells. Tumor weight at 14 days in control rats (those fed standard chow) and CO supplemented was approximately 30 g. Supplementation of the diet with FO significantly reduced tumor growth by 76%. Lactate production (nmol h(-1) mg(-1) protein) from glucose by Walker 256 cells in the group fed regular chow (W) was 381.8 +/- 14.9. Supplementation with coconut fat (WCO) caused a significant reduction in lactate production by 1.6-fold and with fish oil (WFO) by 3.8-fold. Spleen lymphocytes obtained from W and WCO groups had markedly increased lactate production (553 +/- 70 and 635 +/- 150) when compared to non-tumor-bearing rats (similar to 260 +/- 30). FO supplementation reduced significantly the lactate production (297 +/- 50). Gut-associated lymphocytes obtained from W and WCO groups increased lactate production markedly (280 +/- 31 and 276 +/- 25) when compared to non-tumor-bearing rats (similar to 90 +/- 18). FO supplementation reduced significantly the lactate production (168 +/- 14). Lactate production by peritoneal macrophages was increased by tumor burden but there was no difference between the groups fed the various diets. Lifelong consumption of FO protects against tumor growth and modifies glucose metabolism in Walker tumor cells and lymphocytes but not in macrophages. Copyright (C) 2008 John Wiley & Sons, Ltd.

Endogenous Hepatocyte Growth Factor Attenuates Inflammatory Response in Glycerol-Induced Acute Kidney Injury

Background: Hepatocyte growth factor (HGF) is overexpressed after acute kidney injury (AKI). The aim of this study was to evaluate the role of endogenous HGF in the progression of the inflammatory response in glycerol-induced AKI (Gly-AKI) in rats. Methods: Renal and systemic HGF expressions were evaluated during the development of Gly-AKI. Subsequently, the blockade of endogenous HGF was analyzed in rats treated with anti-HGF antibody concomitant to glycerol injection. Apoptosis, cell infiltration and chemokine and cytokine profiles were investigated. Results: We detected an early peak of renal and plasma HGF protein expressions 3 h after glycerol injection. The pharmacological blockade of the endogenous HGF exacerbated the renal impairment, the tubular apoptosis, the renal expression of monocyte chemoattractant protein-1 and the macrophage, CD43+, CD4+ and CD8+ T lymphocytes renal infiltration. The analysis of mRNA expressions of Th1 (t-bet, TNF-alpha, IL-1 beta) and Th2 (gata-3, IL-4, IL-10) cytokines showed a Th1-polarized response in Gly-AKI rats that was aggravated with the anti-HGF treatment. Conclusion: Endogenous HGF attenuates the renal inflammatory response, leukocyte infiltration and Th1 polarization after glycerol injection. The control of cellular immune response may partly explain the protective effect of endogenous HGF in the development of Gly-AKI. Copyright (C) 2008 S. Karger AG, Basel

Inhibitory Signals Mediated by Programmed Death-1 Are Involved With T-Cell Function in Chronic Periodontitis

Background: Inhibitory signals mediated via molecules such as programmed death-1 (PD-1) play a critical role in downmodulating immune responses and maintaining peripheral tolerance. We investigated the involvement of cytokines and PD-1 engagement in mediating the T-cell unresponsiveness to bacterial and ubiquitous antigens in periodontal diseases. Methods: Gingival and peripheral blood samples from healthy individuals and patients with chronic periodontitis were collected and used for the subsequent assays. Leukocytes in the lesion site and blood were evaluated using flow cytometry. The production of interferon-gamma, interleukin-10, and transforming growth factor-P proteins was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of PD-1+cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and PD-1 colocalization. Results: T cells from patients with chronic periodontitis proliferated poorly in response to Aggregatibacter actinomycetem comitans (previously Actinobacillus actinomycetemcomitans) antigen. T-cell unresponsiveness was not associated with imbalanced cytokine production. However, T cells from patients with chronic periodontitis expressed significantly higher levels of PD-1 either upon isolation or after culture with antigens. Moreover, PD-1 blocking did not result in significant T-cell proliferation in cells cultured with phytohemagglutinin or bacterial antigens. The blockade of PD-1 resulted in the increased production of IFN-gamma. In addition, CD4+ and CD8+ T cells expressing PD-1 accumulated in lesions with chronic periodontitis. Conclusion: These data show that PD-1 engagement could be involved in the modulation of IFN-gamma production by T cells in patients with chronic periodontitis. J Periodontol 2009,80:1833-1844.

Donor bone marrow cells play a role in the prevention of accelerated graft rejection induced by semi-allogeneic spleen cells in transplantation

Spleen or spleen plus bone marrow cells from (BALB/c x C57Bl/6)F1 donors were transferred into BALB/c recipients 21 days before skin or cardiac transplantation. Prolonged graft survival was observed on recipients treated with the mixture of donor-derived cells as compared to those treated with spleen cells alone. We evaluated the expression of CD45RB and CD44 by splenic CD4(+) and CD8(+) T cells 7 and 21 days after donor cell transfer. The populations of CD8(+)CD45RB(low) and CD8(+)CD44(high) cells were significantly decreased in mice pre-treated with donor spleen and bone marrow cells as compared to animals treated with spleen cells only, although these cells expanded in both groups when compared to an earlier time-point. No differences were observed regarding CD4+ T cell population when recipients of donor-derived cells were compared. An enhanced production of IL-10 was observed seven days after transplantation in the supernatants of spleen cell cultures of mice treated with spleen and bone marrow cells. Taken together these data suggest that donor-derived bone marrow cells modulate the sensitization of the recipient by semi-allogeneic spleen cells in part by delaying the generation of activated/memory CD8(+) T cells leading to enhanced graft survival. (c) 2007 Elsevier B.V. All rights reserved.

Human endogenous RNAs: Implications for the immunomodulation of Toll-like receptor 3

Toll-like receptors (TLRs), a family of mammalian receptors, are able to recognize nucleic acids. TLR3 recognizes double-stranded (ds)RNA, a product of the replication of certain viruses. Polyinosinic-polycytidylic acid, referred to as poly(I:C), an analog of viral dsRNA, interacts with TLR3 thereby eliciting immunoinflammatory responses characteristic of viral infection or down-regulating the expression of chemokine receptor CXCR4. It is known that dsRNA also directly activates interferon (IFN)-induced enzymes, such as the RNA-dependent protein kinase (PKR). In the present study, the mRNA expression of TLR3, CXCR4, IFN gamma and PKR was investigated in a culture of peripheral blood mononuclear cells (PBMCs) stimulated with poly(I:C) and endogenous RNA from human PBMCs. No cytotoxic effect on the cells or on the proliferation of CD3(+), CD4(+) and CD8(+) cells was observed. TLR3 expression in the PBMCs in the presence of poly(I:C) was up-regulated 9.5-fold, and TLR3 expression in the PBMCs treated with endogenous RNA was down-regulated 1.8-fold (p=0.002). The same trend was observed for IFN gamma where in the presence of poly(I:C) an 8.7-fold increase was noted and in the presence of endogenous RNA a 3.1-fold decrease was observed. In the culture activated with poly(1:C), mRNA expression of CXCR4 increased 8.0-fold and expression of PKR increased 33.0-fold. Expression of these genes decreased in the culture treated with endogenous RNA when compared to the culture without stimulus. Thus, high expression of mRNA for TLR3, IFN gamma, CXCR4 and PKR was observed in the presence of poly(I:C) and low expression was observed in the cells cultured with endogenous RNA. In conclusion, TLR3 may play major physiological roles that are not in the context of viral infection. It is possible that RNA released from cells could contain enough double-stranded structures to regulate cell activation. The involvement of endogenous RNA in endogenous gene expression and its implications in the regulation thereof, are still being studied, and will have significant implications in the future.

PAS-1, an Ascaris suum Protein, Modulates Allergic Airway Inflammation via CD8(+) gamma delta TCR(+) and CD4(+) CD25(+) FoxP3(+) T Cells

We have previously demonstrated that PAS-1, a 200 kDa protein from Ascaris suum, has a potent immunomodulatory effect on humoral and cell-mediated responses induced by APAS-3 (an allergenic protein from A. suum) or unrelated antigens. In this study, we investigated the mechanisms by which PAS-1 is able to induce this effect on an allergic airway inflammation induced by OVA in mice. C57BL/6 mice were adoptively transferred on day 0 with seven different PAS-1-primed cell populations: PAS-1-primed CD19(+) or B220(+) or CD3(+) or CD4(+) or CD8(+) or CD4(+) CD25) or CD4(+) CD25(+) lymphocytes. These mice were immunized twice with OVA and alum by intraperitoneal route (days 0 and 7) and challenged twice by intranasal route (days 14 and 21). Two days after the last challenge, the airway inflammation was evaluated by antibody levels, cellular migration, eosinophil peroxidase levels, cytokine and eotaxin production, and pulmonary mechanical parameters. Among the adoptively transferred primed lymphocytes, only CD4(+) CD25(+), CD8(+) or the combination of both T cells impaired the production of total IgE and OVA-specific IgE and IgG1 antibodies, eosinophilic airway inflammation, Th2-type cytokines (IL-4, IL-5 and IL-13), eotaxin release and airway hyperreactivity. Moreover, airway recruited cells from CD4(+) CD25(+) and CD8(+) T-cell recipient secreted more IL-10/TGF-beta and IFN-gamma, respectively. Moreover, we found that PAS-1 expands significantly the number of CD4(+) CD25(+) FoxP3(+) and CD8(+) gamma delta TCR(+) cells. In conclusion, these findings demonstrate that the immunomodulatory effect of PAS-1 is mediated by these T-cell subsets.

Low CD4(+) T-Cell Levels and B-Cell Apoptosis in Vertically HIV-exposed Noninfected Children and Adolescents

Lymphocyte subsets, activation markers and apoptosis were assessed in 20 HIV-exposed noninfected (ENI) children born to HIV-infected women who were or not exposed to antiretroviral (ARV) drugs during pregnancy and early infancy. ENI children and adolescents were aged 6-18 years and they were compared to 25 age-matched healthy non-HIV-exposed children and adolescents (Control). ENI individuals presented lower CD4(+) T cells/mm(3) than Control group (control: 1120.3 vs. ENI: 876.3; t-test, p=0.030). ENI individuals had higher B-cell apoptosis than Control group (Control: 36.6%, ARV exposed: 82.3%, ARV nonexposed: 68.5%; Kruskal-Wallis, p < 0.05), but no statistical difference was noticed between those exposed and not exposed to ARV. Immune activation in CD4(+) T, CD8(+) T and in B cells was comparable in ENI and in Control children and adolescents. Subtle long-term immune alterations might persist among ENI individuals, but the clinical consequences if any are unknown, and these children require continued monitoring.

CCR5 Mediates Pro-osteoclastic and Osteoclastogenic Leukocyte Chemoattraction

Periodontal disease (PD) progression involves the selective leukocyte infiltration into periodontium, supposedly mediated by the chemokine/chemokine receptor system. In this study, we investigated the role of chemokine receptor CCR5 in the immunoregulation of experimental PD in C57BL/6 (WT) and CCR5KO mice. Aggregatibacter actinomycetem comitans infection triggered the chemoattraction of distinct CCR5+ leukocyte subpopulations (determined by flow cytometry): CCR5+F4/80+ leukocytes, which co-express CD14, CCR2, TNF-alpha, and IL-1 beta, indicative of activated macrophages; and CCR5+CD4+ cells, which co-express CXCR3, IFN-gamma, and RANKL, indicative of Th1 lymphocytes, therefore comprising pro-osteoclastic and osteoclastogenic cell subsets, respectively. CCR5KO mice presented a lower PD severity (lower inflammation and alveolar bone loss) when compared with the WT strain, since the migration of F4/80+, TNF-alpha+, CD4+, and RANKL+ cells specifically decreased due to the lack of CCR5. Also, ELISA analysis demonstrated that the production of TNF-alpha, IL-1 beta, IL-6, IFN-gamma, and RANKL in periodontal tissues was significantly decreased in the CCR5KO strain. The periodontal bacterial load and antimicrobial patterns were unaltered in CCR5KO mice. Our results demonstrate that the chemokine receptor is involved in the migration of distinct leukocyte subpopulations throughout experimental PD, being a potential target for therapeutic intervention in PD.

Chronic colitis associated with HIV infection can be related to intraepithelial infiltration of the colon by CD8+T lymphocytes

Gastrointestinal complications in AIDS patients with diarrhoea are common clinical manifestations, frequently diagnosed by colonoscopy as non-specific colitis. We retrospectively study colon biopsies diagnosed as chronic colitis associated with HIV (CCH). Biopsies were sorted as patients with AIDS (serum CD4 < 200 cell/mm(3)) but without any clear infectious process (n = 12) and patients without HIV infection (n = 24). There are low numbers of CD4+ T lymphocytes in lamina propria of AIDS patients, but CD8+ T populations in this area appear to be similar in all studied groups, regardless of HIV infection or laboratory evidence of a specific agent. We found the clear evidence of CD8+ T cells infiltration in colonic mucosa in HIV patients with microscopic colitis. An imbalance of lymphocyte subpopulations in the colon, both in the lamina propria and epithelium, could result in an intraepithelial CD8 infiltration, involved in the pathogenesis of CCH in AIDS patients.

Changes in histone acetylation and methylation that are important for persistent but not transient expression of CCR4 in human CD4(+) T cells

Although regulation of CXCR3 and CCR4 is related to Th1 and Th2 differentiation, respectively, many CXCR3(+) and CCR4(+) cells do not express IFN-gamma and/or IL-4, suggesting that the chemokine receptor genes might be inducible by mechanisms that are lineage-independent. We investigated the regulation of CXCR3 versus IFNG, and CCR4 versus IL4 in human CD4(+) T cells by analyzing modifications of histone H3. In naive cord-blood cells, under nonpolarizing conditions not inducing IL4, CCR4 was induced to high levels without many of the activation-associated changes in promoter histone H3 found for both IL4 and CCR4 in Th2 cells. Importantly, CCR4 expression was stable in Th2 cells, but fell in nonpolarized cells after the cells were rested; this decline could be reversed by increasing histone acetylation using sodium butyrate. Patterns of histone H3 modifications in CXCR3(+) CCR4(-) and CXCR3(-) CCR4(+) CD4(+) T-cell subsets from adult blood matched those in cells cultured under polarizing conditions in vitro. Our data show that high-level lineage-independent induction of CCR4 can occur following T-cell activation without accessibility-associated changes in histone H3, but that without such changes expression is transient rather than persistent.

Effect of Fish Oil Supplementation for Two Generations on Changes of Lymphocyte Function Induced by Walker 256 Cancer Cachexia in Rats

Fish oil supplementation has been shown to improve the cachectic state of tumor-bearing animals and humans. Our previous study showed that fish oil supplementation (1 g per kg body weight per day) for 2 generations had anticancer and anticachetic effects in Walker 256 tumor-bearing rats as demonstrated by reduced tumor growth and body weight loss and increased food intake and survival. In this study, the effect of fish oil supplementation for 2 generations on membrane integrity, proliferation capacity, and CD4/CD8 ratio of lymphocytes isolated from mesenteric lymph nodes, spleen, and thymus of Walker 256 tumor-bearing animals was investigated. We also determined fish oil effect on plasma concentration and ex vivo production of cytokines [tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6, and IL-10]. Lymphocytes from thymus of tumor-bearing rats presented lower viability, but this change was abolished by fish oil supplementation. Tumor growth increased proliferation of lymphocytes from all lymphoid organs, and fish oil supplementation abolished this effect. Ex vivo production of TNF-alpha and IL-6 was reduced in supplemented animals, but IL-4 and IL-10 secretion was stimulated in both nontumor and tumor-bearing rats. IL-10 and IFN-gamma plasma levels was also decreased in supplemented animals. These results suggest that the anticachetic effects of fish oil supplementation for a long period of time (2 generations) in Walker 256 tumor-bearing rats may be associated to a decrease in lymphocyte function as demonstrated by reduced viability, proliferation capacity, and cytokine production.

Effect of doxorubicin on cytokine production by lymphocytes and the Th1/Th2 balance

Doxorubicin (DOXO) is a potent chemotherapeutic used mainly against solid tumours; however, it has several side effects that can limit its clinical use. On the other hand, the effect of DOXO upon lymphocyte function is controversial. Some studies demonstrate that DOXO administration in vitro suppresses T-cell activation, while the cellular function has been shown to increase in vitro. The objective of this study was to investigate the effect of DOXO on lymphocyte cytokine production in rats. The animals were divided into: SAL (control, n = 10) and DOX (DOXO treated, n = 10). The DOX group received only one DOXO dose at 15 kg Kg(-1) by intraperitoneal injection. Forty-eight hours after DOXO administration, the animals were killed by decapitation. IL-2 production was significantly enhanced (p < 0.05) in lymphocytes from rats treated with DOXO (169.17 +/- 21.73 pg mL 10(5) cell) as compared to cells from SAL (45.92 +/- 10.53 pg mL 10(5) cell). The administration of DOXO decreased (<0.05) IL-4 production in the DOXO group (29.85 +/- 13.09 pg mL 10(5) cell) relative to the SAL group (75.08 +/- 15.31 pg mL 10(5) cell). The IL-2/IL-4 ratio was higher (<0.05) in the DOX group (5.99 +/- 0.44), as compared to SAL group (0.73 +/- 0.12). In conclusion, our results suggest that a dose of DOXO promotes an alteration in the Th1/Th2 balance, promoting a shift towards a Th1-dominant cytokine response. (C) 2010 Elsevier Masson SAS. All rights reserved.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.