S. De La Barrera

IL‐10 down‐regulates costimulatory molecules on Mycobacterium tuberculosis‐pulsed macrophages and impairs the lytic activity of CD4 and CD8 CTL in tuberculosis patients

Activation of T cells requires both TCR‐specific ligation and costimulation through accessory molecules during T cell priming. IFNγ is a key cytokine responsible for macrophage activation during Mycobacterium tuberculosis (Mtb) infection while IL‐10 is associated with suppression of cell mediated immunity in intracellular infection. In this paper we evaluated the role of IFNγ and IL‐10 on the function of cytotoxic T cells (CTL) and on the modulation of costimulatory molecules in healthy controls and patients with active tuberculosis (TB). γ‐irradiated‐Mtb (i‐Mtb) induced IL‐10 production from CD14+ cells from TB patients. Moreover, CD3+ T cells of patients with advanced disease also produced IL‐10 after i‐Mtb stimulation. In healthy donors, IL‐10 decreased the lytic activity of CD4+ and CD8+ T cells whereas it increased γδ‐mediated cytotoxicity. Furthermore, we found that the presence of IL‐10 induced a loss of the alternative processing pathways of antigen presentation along with a down‐regulation of the expression of costimulatory molecule expression on monocytes and macrophages from healthy individuals. Conversely, neutralization of endogenous IL‐10 or addition of IFNγ to either effector or target cells from TB patients induced a strong lytic activity mediated by CD8+ CTL together with an up‐regulation of CD54 and CD86 expression on target cells. Moreover, we observed that macrophages from TB patients could use alternative pathways for i‐Mtb presentation. Taken together, our results demonstrate that the presence of IL‐10 during Mtb infection might contribute to mycobacteria persistence inside host macrophages through a mechanism that involved inhibition of MHC‐restricted cytotoxicity against infected macrophages.

Specific lytic activity against mycobacterial antigens is inversely correlated with the severity of tuberculosis

The ability of peripheral blood mononuclear cells (PBMC) from patients with active tuberculosis to display cytotoxic responses against autologous Mycobacterium tuberculosis (Mtb)‐pulsed macrophages was evaluated. Non‐MHC restricted cell‐dependent lytic activity was observed in ex vivo effector cells from tuberculosis patients and was mediated mainly by CD3+γδ TCR+ T (γδ T) cells bearing CD56 and/or CD16 molecules. MHC‐restricted and non‐MHC restricted cytotoxic T cells (CTL) were differentially expanded upon stimulation with Mtb in tuberculosis patients and normal controls (N). Class‐I restricted CD8+ CTL and class‐II restricted CD4+ CTL were generated in PPD+N and to a lesser extent in PPD–N. Mtb‐stimulated effector cells from tuberculosis patients became progressively non‐MHC restricted CD4–CD8–γδ T cells, while lytic activity of CD4+ and CD8+CTL decreased gradually as the disease became more severe. On the other hand, target cells were lysed by ex vivo cells from tuberculosis patients through the Fas‐FasL and perforin pathways. Mtb‐induced CD4+ CTL from tuberculosis patients and N controls preferentially employed the Fas‐FasL mechanism. Mtb‐induced CD8+ CTL effector cells from patients used the perforin‐based mechanism while cells from N controls also used the Fas‐FasL pathway. While Mtb‐induced γδ CTL from patients and PPD–N employed the latter mechanism cells from PPD+N individuals also used the perforin pathway. It can be concluded that shifts in the CTL response and the cytolytic mechanisms take place as the pulmonary involvement becomes more severe.

CD4+CD25highforkhead box protein 3+ regulatory T lymphocytes suppress interferon‐γ and CD107 expression in CD4+ and CD8+ T cells from tuberculous pleural effusions

Tuberculous pleural effusion is characterized by a T helper type 1 (Th1) profile, but an excessive Th1 response may also cause tissue damage that might be controlled by regulatory mechanisms. In the current study we investigated the role of regulatory T cells (Treg) in the modulation of Th1 responses in patients with tuberculous (TB) pleurisy. Using flow cytometry we evaluated the proportion of Treg (CD4+CD25highforkhead box protein 3+), interferon (IFN)‐γ and interleukin (IL)‐10 expression and CD107 degranulation in peripheral blood (PB) and pleural fluid (PF) from patients with TB pleurisy. We demonstrated that the proportion of CD4+CD25+, CD4+CD25highFoxP3+ and CD8+CD25+ cells were increased in PF compared to PB samples. Mycobacterium tuberculosis stimulation increased the proportion of CD4+CD25low/negIL‐10+ in PB and CD4+CD25low/negIFN‐γ+ in PF; meanwhile, CD25high mainly expressed IL‐10 in both compartments. A high proportion of CD4+CD107+ and CD8+CD107+ cells was observed in PF. Treg depletion enhanced the in‐vitro M. tuberculosis‐induced IFN‐γ and CD4+ and CD8+ degranulation responses and decreased CD4+IL‐10+ cells in PF. Our results demonstrated that in TB pleurisy Treg cells effectively inhibit not only IFN‐γ expression but also the ability of CD4+ and CD8+ cells to degranulate in response to M. tuberculosis.

CD3 expression distinguishes two γδT cell receptor subsets with different phenotype and effector function in tuberculous pleurisy

Tuberculous pleurisy is a naturally occurring site of Mycobacterium tuberculosis (Mtb) infection. Herein, we describe the expression of activation, natural killer (NK) and cell migration markers, as well as effector functions from γδT cells in peripheral blood (PB) and pleural effusion (PE) from tuberculosis patients (TB). We observed a decreased percentage of circulating γδT from TB patients and differential expression of NK as well as of chemokine receptors on PB and PE. Two subsets of γδT cells were differentiated by the CD3/γδT cell receptor (γδTCR) complex. The γδTCRlow subset had a higher CD3 to TCR ratio and was enriched in Vδ2+ cells, whereas most Vδ1+ cells belonged to the γδTCRhigh subset. In PB from TB, most γδTCRhigh were CD45RA+CCR7‐ and γδTCRlow were CD45RA+/−CCR7+CXCR3+. In the pleural space the proportion of CD45RA‐CCR7+CXCR3+ cells was higher. Neither spontaneous nor Mtb‐induced interferon (IFN)‐γ production was observed in PB‐γδT cells from TB; however, PE‐γδT cells showed a strong response. Both PB‐ and PE‐γδ T cells expressed surface CD107a upon stimulation with Mtb. Notably, PE‐γδTCRlow cells were the most potent effector cells. Thus, γδT cells from PB would acquire a further activated phenotype within the site of Mtb infection and exert full effector functions. As γδT cells produce IFN‐γ within the pleural space, they would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a T helper type 1 profile.

IL‐9 promotes anti‐Mycobacterium leprae cytotoxicity: involvement of IFNγ

Interleukin 9 (IL‐9) is a T‐cell derived factor preferentially expressed by CD4+ Th2 cells and it has been characterized both in human and murine systems. It is a pleiotropic cytokine with multiple functions on cells of the lymphoid, myeloid and mast cell lineages, as well as on lung epithelial cells. Other activities described for IL‐9 support its contribution to asthma and its important role in helminthic infections, where a Th2 response can be protective and IL‐9 enhances resistance or is responsible for elimination of the nematode. Nevertheless, until recently there were no studies on its role in bacterial infections in man. We have demonstrated that cytokines can modulate the specific cytotoxicity generation in peripheral blood mononuclear cells from leprosy patients and normal controls. In the present report we studied the effect of IL‐9 in this experimental model. Our results indicate that IL‐9 can counteract the negative effect mediated by IL‐4 on the generation of M. leprae‐induced cytotoxic T lymphocytes. Moreover, it can increase this lytic activity in controls and enhance the stimulatory effect of IL‐2 or IL‐6 in cells from leprosy patients and controls. IL‐9 is also able to revert the inhibitory effect of IL‐10 and IL‐13 on the M. leprae‐induced cytotoxic activity. Although the exact mechanism of action of IL‐9 remains to be determined, interferon gamma seems to be required for the effect of IL‐9 in this experimental model. These data suggest that IL‐9 may have an atypical Th2 behaviour and play a role in the modulation of the immune response to mycobacterial infections.

Lack of cytotoxic activity against Mycobacterium leprae 65-kD heat shock protein (hsp) in multibacillary leprosy patients

Cytotoxic T cells play an important role in host defence mechanisms, as well as in the immunopathology of leprosy. In this study, we evaluated whether Mycobacterium leprae hsp18, hsp65 and Myco. tuberculosis hsp71 could induce cytotoxic T cell activity against autologous macrophages pulsed with these hsp. Paucibacillary (PB) patients and normal controls generated more effector cells than multibacillary (MB) patients with all three hsp tested. There was no cross‐reactivity between any of the hsp tested. Mycobacterium leprae hsp65 induced cytotoxic responses only in those MB patients undergoing an erythema nodosum leprosum (ENL) episode. Although hsp65 and hsp18 induced similar proliferation in MB patients, a high proportion of these patients did not generate cytotoxic effector cells in response to hsp65. Hence, those T cells reacting to hsp65 may play an important role in the control of Myco. leprae infection.

Immune complexes (IC) down‐regulate the basal and interferon‐γ‐induced expression of MHC Class II on human monocytes

The interaction of Fc receptors for IgG (FcγRs) on monocytes/macrophages with immune complexes (IC) triggers regulatory and effector functions. Previous studies have shown that FcγR–IC interactions inhibit the IFN‐γ‐induced expression of MHC class II in murine macrophages. However, the mechanism(s) responsible for these effects have not been elucidated. In addition, whether this IC‐dependent effect also occurs in human cells is not known. Taking into account the fact that IC and IFN‐γ are frequently found in infections and autoimmune disorders, together with the crucial role MHC class II molecules play in the regulation of immune response, we explored the effect and mechanism of IC‐induced MHC class II down‐regulation in human peripheral blood mononuclear cells (PBMC). This effect was studied either in the presence or absence of IFN‐γ. We demonstrate that IC exert a drastic inhibition of basal and IFN‐γ‐induced expression of MHC class II on human monocytes. This effect was mediated through the interaction of IC with both FcγRI and FcγRII. Moreover, similar results were obtained using supernatants from IC‐treated PBMC. The IC‐induced down‐regulation of MHC class II is abrogated by pepstatin and phosphoramidon, supporting the role of aspartic protease(s) and metalloprotease(s) in this process. In parallel with MHC class II expression, antigen presentation was markedly inhibited in the presence of IC.

NK cells modulate the cytotoxic activity generated by Mycobacterium leprae -hsp65 in leprosy patients: role of IL-18 and IL-13

Protection against intracellular pathogens such as Mycobacterium leprae is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages. In the present report we evaluated the role of IL‐18 and IL‐13, two cytokines that can influence NK cell activity, in the generation of M. leprae‐derived hsp65‐cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) of leprosy patients. We demonstrated that IL‐18 modulates hsp65‐induced CTL generation and collaborates with IL‐12 for this effect. In paucibacillary (PB) patients and normal controls (N) depletion of NK cells reduces the cytolytic activity. Under these conditions, IL‐12 cannot up‐regulate this CTL generation, while, in contrast, IL‐18 increases the cytotoxic activity both in the presence or absence of NK cells. IL‐13 down‐regulates the hsp65‐induced CTL generation and counteracts the positive effect of IL‐18. The negative effect of IL‐13 is observed in the early stages of the response, suggesting that this cytokine affects IFNγ production by NK cells. mRNA coding for IFNγ is induced by IL‐18 and reduced in the presence of IL‐13, when PBMC from N or PB patients are stimulated with hsp65. Neutralization of IL‐13 in PBMC from multibacillary (MB) leprosy patients induces the production of IFNγ protein by lymphocytes. A modulatory role on the generation of hsp65 induced CTL is demonstrated for IL‐18 and IL‐13 and this effect takes place through the production of IFNγ.

Toll-Like Receptors in Human Infectious Diseases

Toll-like receptors (TLRs) have emerged as critical players in immunity. They are capable of sensing organisms ranging from protozoa to bacteria, fungi or viruses upon detection of the pathogen as well as recognizing endogenous ligands, and triggering transduction pathways. Following activation of the innate immune system, strong inflammatory signals are generated inducing inflammation and activation of the adaptive immune response. However, the deregulation of TLRs signaling pathways may be conducive to the pathogenesis of many infectious diseases. Therefore, innate and adaptive immunity are not simply sequential and complementary mechanisms of resistance to pathogen, they regulate each other through cellular contacts and the secretion of soluble mediators. Herein, we summarize recent findings on TLRs signaling in infectious diseases and how pathogens have developed strategies to evade these pathways. In this context, a potential modulation of the innate immune response could have therapeutic benefit through the development of new drugs as well as vaccination strategies to be employed in infectious diseases.