Cutaneous Site Of Infection Research Articles

The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Signal transducer and activator of transcription 1 in T cells plays an indispensable role in immunity to Leishmania major by mediating Th1 cell homing to the site of infection

The signal transducer and activator of transcription 1 (STAT1) signaling pathway mediates the biological functions of IFN-gamma. We have previously shown that the STAT1 pathway is indispensable for host resistance against Leishmania major infection. In this study, we examined the role of STAT1 in lymphocytes and specifically CD4(+) and CD8(+) T cells in mediating immunity against L. major by transferring T cells from wild-type (WT) and STAT1(-/-) C57BL/6 mice into Rag2(-/-) C57BL/6 mice. Rag2(-/-) mice reconstituted with unfractionated STAT1(-/-) splenocytes (B cells and T cells) failed to mount an efficient Th1 response after L. major infection, produced more IL-4, and developed large lesions full of parasites. In contrast, Rag2(-/-) mice reconstituted with WT (STAT1(+/+)) splenocytes mounted a Th1 response and developed self-resolving lesions. Studies using Rag2(-/-) recipients that received a combination of purified CD4(+) and CD8(+) T cells from WT or STAT1(-/-) mice revealed that STAT1 deficiency in CD4(+) T cells, but not in CD8(+) T cells, leads to development of chronic, nonhealing lesions and systemic dissemination of parasites into the spleen after L. major infection. Further studies using Rag2(-/-) recipients of WT Thy1.1(+) and STAT1(-/-) Thy1.2(+) T cells showed that STAT1 in CD4(+) T cells was not required for Th1 differentiation during L. major infection. However, it was critical for up-regulation of CXCR3 on CD4(+) T cells and their migration to the regional lymph node and the cutaneous site of infection. Together, these studies indicate that the STAT1 pathway in CD4(+) T cells plays a critical role in immunity against L. major by controlling the migration of Th1 cells to the site of infection rather than their generation. Further, they reveal an essential role for CD4(+) T cell STAT1 in preventing systemic dissemination of L. major infection.

Selective deficiency of STAT1 in CD4+ T cells enhances susceptibility to Leishmania major infection not by preventing induction of Th1 response, but by impairing CD4+ T cell homing (44.26)

Abstract We previously reported that STAT1 signaling is indispensable for host resistance against Leishmania major. In this study, we examined the role of STAT1 in lymphocytes as well as CD4+ versus CD8+ T cells in mediating immunity to L. major by performing adoptive transfers of T cells from WT and STAT1-/- C57BL/6 mice to Rag2-/- C57BL/6 mice. Rag2-/- mice reconstituted with STAT1-/- splenocytes (B cells and T cells) failed to mount an efficient Th1 response following L. major infection, produced more IL-4 and developed large lesions full of parasites. In contrast, Rag2-/- mice reconstituted with WT (STAT1+/+) splenocytes mounted a Th1 response and developed self-resolving lesions. Studies using Rag2-/- recipients that received a combination of purified CD4+ and CD8+ T cells from WT or STAT1-/- mice revealed that STAT1 in CD4+, but not CD8+ T cells was indispensable for immunity against L. major. Further studies using Thy1.1+ congenic recipients receiving WT or STAT1 Thy1.2+ CD4+ cells showed that STAT1 in CD4+ T cells was not required for Th1 differentiation and IFN-γ production during L. major infection, but was critical for migration to regional lymph nodes and the cutaneous site of infection. Together these studies indicate that STAT1 signaling in CD4+ T cells plays a critical role in immunity against L. major by controlling migration of Th1 cells to the site of infection rather than their generation. Research Support: NIH

CD4+T Cells Play a Dominant Role in Protection against New World Leishmaniasis Induced by Vaccination with the P-4 Amastigote Antigen

Immunodepletion studies of P-4-vaccinated mice indicate that CD4+ and not CD8+ T cells are critical for protection against Leishmania pifanoi (Leishmania mexicana complex). Although a moderate CD8+ T-cell response is elicited by vaccination, CD4+ T cells are the dominant responding population in vitro and at the cutaneous site of infection. These protective T cells produce gamma interferon (IFN-gamma), macrophage migration inhibitory factor (MIF), and tumor necrosis factor/lymphotoxin (TNF/LT), each of which significantly contributed to intracellular parasite destruction in vitro. These results indicate that a singular CD4+ T-cell response (IFN-gamma, MIF, and/or LT/TNF) can provide protection against New World cutaneous leishmaniasis.

Perforin and gamma interferon are critical CD8+ T-cell-mediated responses in vaccine-induced immunity against Leishmania amazonensis infection.

Previous studies have demonstrated that protection against New World leishmaniasis caused by Leishmania amazonensis can be elicited by immunization with the developmentally regulated Leishmania amastigote antigen, P-8. In this study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved. T-cell subset depletion experiments clearly indicate that elicitation of CD8(+) (as well as CD4(+)) effector responses is required for protection. Further, mice lacking beta(2)-microglobulin (and hence deficient in major histocompatibility complex class I antigen presentation) were not able to control a challenge infection after vaccination, indicating an essential protective role for CD8(+) T effector responses. Analysis of the events ongoing at the cutaneous site of infection indicated a changing cellular dynamic involved in protection. Early postinfection in protectively vaccinated mice, a predominance of CD8(+) T cells, secreting gamma interferon (IFN-gamma) and expressing perforin, was observed at the site of infection; subsequently, activated CD4(+) T cells producing IFN-gamma were primarily found. As protection correlated with the ratio of total IFN-gamma-producing cells (CD4(+) and CD8(+) T cells) to macrophages found at the site of infection, a role for IFN-gamma was evident; in addition, vaccination of IFN-gamma-deficient mice failed to provide protection. To further assess the effector mechanisms that mediate protection, mice deficient in perforin synthesis were examined. Perforin-deficient mice vaccinated with the P-8 antigen were unable to control infection. Thus, the elicitation of CD8(+) T cell effector mechanisms (perforin, IFN-gamma) are clearly required in the protective immune response against L. amazonensis infection in vaccinated mice.

Leishmania pifanoi pathogenesis: selective lack of a local cutaneous response in the absence of circulating antibody.

Recently, a role for B cells in the pathogenesis associated with infection by Leishmania (Leishmania mexicana complex and L. donovani) has been established. In the case of L. mexicana complex parasites (L. mexicana, L. pifanoi, and L. amazonensis), a critical role for immunoglobulin G-mediated mechanisms for the amastigote stage in the host is evident; however, the immunological mechanisms involved remain to be established. In vitro analysis of the kinetics of parasite uptake by macrophages failed to indicate a major effect of antibody opsonization. Given the importance of CD4(+) T cells in the development of disease caused by these parasites, the possibility that the lack of pathogenesis was due to the lack of development of an immune response at the local site (draining lymph node and/or cutaneous site) was explored. Interestingly, the level of CD4(+)-T-cell activation (proliferation and cytokine) in draining lymph nodes from mice lacking circulating antibody (resistant) was found to be comparable to that in nodes from wild-type mice (susceptible) at 2, 5, and 10 weeks postinfection. However, antibody-deficient animals had markedly reduced numbers of monocytes and lymphocytes recruited or retained at the site of cutaneous infection in comparison to wild-type mice, indicating a selective impairment in the local cutaneous immune response. In vitro antigen presentation studies employing tissue-derived (opsonized) amastigotes demonstrated that L. pifanoi-infected FcR(-/-) macrophages, in contrast to comparably infected wild-type cells, failed to activate Leishmania antigen-specific T lymphocytes. These data, taken together, suggest that one possible mechanism for the role of antibody in pathogenesis may be to mediate parasite uptake and regulate the immune response at the local cutaneous site of infection.

The migration and attrition of Strongyloides ratti in naive and sensitized rats

Compressed organ autoradiography has been utilized to study the migration of the homogonic strain of Strongyloides ratti from the site of skin penetration to the gut. This transit is characterized by rapid disappearance of parasites from the cutaneous site of infection, followed by vascular dispersal throughout all organs investigated. For the first time, parasite migration in both naive and previously sensitized hosts is compared; the principal stations of migration are the same in the two groups although there is a quantitative difference in accumulation of parasites with time in these stations. Parasite attrition occurs in both groups of animals; however, in naive rats it is not manifest until day 20-25 post challenge, whereas in sensitized rats immune elimination occurs as early as 48 h post challenge.

Antigen-specific immune-suppressor factor in herpes simplex virus type 2 infections of UV B-irradiated mice

UV B-irradiation (280 to 320 nm) of mice at the site of cutaneous infection with herpes simplex virus type 2 (HSV-2) induced suppressor T-cell circuits that decreased HSV-2-induced proliferative responses of HSV-2-immune lymph node cells. Adoptive transfer experiments indicated that splenocytes from UV B-irradiated HSV-2-infected animals contain L3T4+ cells that suppress proliferative responses in vivo, consistent with suppressor inducer cells. However, following in vitro culture of the splenocytes with HSV-2 antigen, the proliferation of immune lymph node cells was inhibited by Lyt2+ suppressor T cells, consistent with antigen-induced suppressor effector cells. Antigen-specific and nonspecific suppressor factors were fractionated from supernatants of HSV-2-stimulated spleen cells by molecular-sieve chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the Sephadex fraction that contained the antigen-specific suppressor factor, in the presence or absence of 2-mercaptoethanol, defined a 115-kilodalton protein consisting of two disulfide-bound components with molecular sizes of 70 and 52 kilodaltons. The implications of these results with respect to the regulation of HSV-induced cell-mediated immunity following UV B-irradiation are discussed.