Lucia E. Rosas

Cutting Edge: The SLAM Family Receptor Ly108 Controls T Cell and Neutrophil Functions

Ly108, a glycoprotein of the signaling lymphocytic activation molecule family of cell surface receptors expressed by T, B, NK, and APCs has been shown to have a role in NK cell cytotoxicity and T cell cytokine responses. In this study, we describe that CD4+ T cells from mice with a targeted disruption of exons 2 and 3 of Ly108 (Ly108ΔE2+3) produce significantly less IL-4 than wild-type CD4+ cells, as judged by in vitro assays and by in vivo responses to cutaneous infection with Leishmania mexicana. Surprisingly, neutrophil functions are controlled by Ly108. Ly108ΔE2+3 mice are highly susceptible to infection with Salmonella typhimurium, bactericidal activity of Ly108ΔE2+3 neutrophils is defective, and their production of IL-6, IL-12, and TNF-α is increased. The aberrant bactericidal activity by Ly108ΔE2+3 neutrophils is a consequence of severely reduced production of reactive oxygen species following phagocytosis of bacteria. Thus, Ly108 serves as a regulator of both innate and adaptive immune responses.

Macrophage Migration Inhibitory Factor Plays a Critical Role in Mediating Protection against the Helminth Parasite Taenia crassiceps

ABSTRACT To determine the role of endogenous migration inhibitory factor (MIF) in regulation of immune response during murine cysticercosis caused by the helminth parasite Taenia crassiceps, we analyzed the course of T. crassiceps infection in MIF−/− BALB/c mice. MIF−/− mice were highly susceptible to T. crassiceps and developed significantly higher parasite loads compared to similarly infected MIF+/+ mice. Throughout the course of infection, Taenia crassiceps soluble antigen-stimulated spleen cells from both MIF+/+ and MIF−/− mice produced significant and comparable levels of interleukin-4 (IL-4), but those from MIF−/− mice produced significantly more IL-13, as well as gamma interferon (IFN-γ), suggesting that the susceptibility of MIF−/− mice to T. crassiceps was not due to the lack of IFN-γ production. Interestingly, low levels of both total and specific immunoglobulin G2a were observed in MIF−/− cysticercotic mice despite the high IFN-γ levels; in addition, peritoneal macrophages obtained from T. crassiceps-infected MIF−/− mice at different time points failed to respond efficiently to stimulation in vitro with lipopolysaccharide plus IFN-γ and produced significantly lower levels of IL-12, tumor necrosis factor alpha, and NO compared to those from MIF+/+ mice. These findings demonstrate that MIF plays a critical role in mediating protection against T. crassiceps in vivo. Moreover, these findings also suggest that impaired macrophage function rather than the lack of Th1 development may be responsible for mediating susceptibility to T. crassiceps.

PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous leishmaniasis

To compare the roles of programmed death 1 ligand 1 (PD‐L1) and PD‐L2 in regulating immunity to infection, we investigated responses of mice lacking PD‐L1 or PD‐L2 to infection with Leishmania mexicana. PD‐L1–/– and PD‐L2–/– mice exhibited distinct disease outcomes following infection with L. mexicana. In comparison to susceptible WT mice, PD‐L1–/– mice showed resistance to L. mexicana, as demonstrated by reduced growth of cutaneous lesions and parasite burden. In contrast, PD‐L2–/– mice developed exacerbated disease with increased parasite burden. Host resistance to L. mexicana is partly associated with the development of a Th1 response and down‐regulation of the Th2 response. Both PD‐L1–/– and PD‐L2–/– mice produced levels of IFN‐γ similar to WT mice. However, the development of IL‐4‐producing cells was reduced in PD‐L1–/– mice, demonstrating a role for PD‐L1 in regulating Th cell differentiation. This inadequate Th2 response may explain the increased resistance of PD‐L1–/– mice. Although no alterations in Th1/Th2 skewing were observed in PD‐L2–/– mice, PD‐L2–/– mice exhibited a marked increase in L. mexicana‐specific antibody production. Increased Leishmania‐specific IgG production may suppress the healing response through FcγR ligation on macrophages. Taken together, our results demonstrate that PD‐L1 and PD‐L2 have distinct roles in regulating the immune response to L. mexicana.

CXCR3–/– mice mount an efficient Th1 response but fail to control Leishmania major infection

Chemokines play a critical role in recruitment of leukocytes to the site of infection, which is essential for host defense. We analyzed the role of CXC chemokine receptor 3 (CXCR3) in the control of cutaneous leishmaniasis using CXCR3–/– C57BL/6 mice. We found that Leishmania major‐infected CXCR3–/– mice mount an efficient Th1 response as evident by markedly increased serum levels of Th1‐associated IgG2a and significant production of IFN‐γ and IL‐12 by the draining lymph node cells, restrict systemic spread of infection, but fail to control parasite replication at the site of infection and develop chronic non‐healing lesions. Furthermore, the inability of CXCR3–/– mice to control cutaneous L. major growth was associated with fewer CD4+ and CD8+ T cells and significantly lower levels of IFN‐γ in their lesions as compared to CXCR3+/+ mice. These results demonstrate that CXCR3 plays a critical role in the host defense against cutaneous leishmaniasis caused by L. major. Furthermore, they also suggest that the susceptibility of CXCR3–/– mice to L. major is due to impaired CD4+ and CD8+ T cell trafficking and decreased production of IFN‐γ at the site of infection rather than to their inability to mount a parasite‐specific Th1 response.

A STAT4-dependent Th1 response is required for resistance to the helminth parasite Taenia crassiceps

ABSTRACT To determine the role of STAT4-dependent Th1 responses in the regulation of immunity to the helminth parasite Taenia crassiceps, we monitored infections with this parasite in resistant mice lacking the STAT4 gene. While T. crassiceps-infected STAT4+/+ mice rapidly resolved the infection, STAT4−/− mice were highly susceptible to infection and displayed large parasite loads. Moreover, the inability of STAT4−/− mice to control the infection was associated with the induction of an antigen-specific Th2-type response characterized by significantly higher levels of Th2-associated immunoglobulin G1 (IgG1) and total IgE as well as interleukin-4 (IL-4), IL-10, and IL-13 than those in STAT4+/+ mice, who produced significantly more gamma interferon. Furthermore, early after infection, macrophages from STAT4−/− mice produced lower levels of the pro-inflammatory cytokines IL-12, tumor necrosis factor alpha, IL-1β, and nitric oxide (NO) than those from STAT4+/+ mice, suggesting a pivotal role for macrophages in mediating protection against cysticercosis. These findings demonstrate a critical role for the STAT4 signaling pathway in the development of a Th1-type immune response that is essential for mediating protection against the larval stage of T. crassiceps infection.

Cutting Edge: STAT1 and T-bet Play Distinct Roles in Determining Outcome of Visceral Leishmaniasis Caused by Leishmania donovani

T-bet and STAT1 regulate IFN-γ gene transcription in CD4+ T cells, which mediate protection against Leishmania. Here we show that T-bet and STAT1 are required for the induction of an efficient Th1 response during Leishmania donovani infection, but they play distinct roles in determining disease outcome. Both STAT1−/− and T-bet−/− mice failed to mount a Th1 response, but STAT1−/− mice were highly resistant to L. donovani and developed less immunopathology, whereas T-bet−/− mice were highly susceptible and eventually developed liver inflammation. Adoptive cell transfer studies showed that RAG2−/− recipients receiving STAT1+/+ or STAT1−/− T cells developed comparable liver pathology, but those receiving STAT1−/− T cells were significantly more susceptible to infection. These unexpected findings reveal distinct roles for T-bet and STAT1 in mediating host immunity and liver pathology during visceral leishmaniasis.

Development of protective immunity against cutaneous leishmaniasis is dependent on STAT1-mediated IFN signaling pathway.

Although STAT1‐dependent signaling mediates biological functions of IFN‐α/β and IFN‐γ, recent reports indicate that STAT1‐independent IFN signaling also regulates expression of several genes. To determine the roles of STAT1‐dependent and ‐independent IFN signaling in the regulation of immunity during cutaneous leishmaniasis, we studied the course of Leishmania major infection in resistant C57BL/6 mice lacking the STAT1 gene. While L. major‐infected STAT1+/+ mice resolved their lesions, STAT1–/– mice developed large lesions containing significantly more parasites. Moreover, the inability of STAT1–/– mice to control L. major infection was due to the lack of Th1 development associated with reduced production of IL‐12, IFN‐γ and nitric oxide. Although STAT1–/– mice produced more IL‐4 and total IgE than STAT1+/+ mice later during infection, these differences were not significant. Nevertheless, at these time points lymph node cells from STAT1–/– mice produced significantly more IL‐10. Finally, STAT1–/– mice were also susceptible to low dose L. major infection. Thesefindings demonstrate that STAT1‐mediated IFN signaling is indispensable for the development of protective immunity against cutaneous L. major infection. Moreover, they also suggest that the protective role of STAT1‐mediated signaling is due to its ability to induce Th1 development during infection with this parasite.

Lack of CXCR3 Delays the Development of Hepatic Inflammation but Does Not Impair Resistance to Leishmania donovani

CXC chemokine receptor 3 (CXCR3) ligands CXCL9 and CXCL10 are produced at high levels in mice and humans infected with Leishmania donovani, but their contribution to host resistance against L. donovani is not clear. Here, using CXCR3(-/-) mice, we demonstrate that, although CXCR3 regulates early immune cell trafficking and hepatic inflammation during L. donovani infection, it is not essential for immunity against L. donovani, unlike L. major. CXCR3(-/-) C57BL/6 mice show a delayed onset of hepatic inflammation and granuloma formation after L. donovani infection. However, they mount an efficient T helper cell type 1 response, recruit T cells to the liver, and control parasite growth as efficiently as do CXCR3(+/+) C57BL/6 mice.

CC chemokine receptor 1 enhances susceptibility to Leishmania major during early phase of infection

CC chemokine receptor 1 (CCR1) is expressed on the surfaces of monocytes, lymphocytes, neutrophils and eosinophils. CC chemokine receptor 1 not only regulates leucocyte chemotaxis, but also plays a role in the regulation of Th1/Th2 cytokine responses. To determine the role of CCR1 in regulation of immune response during Leishmania major infection, we analysed the course of cutaneous L. major infection in CCR1‐deficient C57BL/6 mice (CCR1−/−) and compared with similarly infected wild‐type mice (CCR1+/+). Following L. major infection, CCR1−/− mice developed significantly smaller lesions containing fewer parasites than CCR1+/+ mice. Furthermore, the severity of the inflammation as assessed by the degree of leucocyte infiltration at the site of infection was similar in CCR1+/+ and CCR1−/− mice. Although both groups developed significant antibody responses following L. major infection, CCR1−/− mice produced significantly lower IgE. On day 20 postinfection, LmAg‐stimulated lymph node cells from L. major‐infected CCR1+/+ and CCR1−/− mice produced comparable levels of IL‐12 and IFN‐γ, but those from CCR1−/− mice produced significantly less IL‐4 and IL‐10. By day 70, lymph node cells from both CCR1+/+ and CCR1−/− mice produced significant amounts of IL‐12 and IFN‐γ but low IL‐4. At both time points, the draining lymph nodes from CCR1+/+ and CCR1−/− mice contained similar number of leucocytes. These results demonstrate that CCR1 plays a role in pathogenesis of cutaneous L. major infection. Moreover, they also indicate that CCR1 exacerbates L. major infection in C57BL/6 mice by up‐regulating Th2‐like response rather than inhibiting Th1 development or/and influencing leucocyte chemotaxis.

A proposed role for Leishmania major carboxypeptidase in peptide catabolism

Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids--the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes--the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.