Landi V. C. Guillermo

The Fas death pathway controls coordinated expansions of Type 1 CD8 and Type 2 CD4 T cells in Trypanosoma cruzi infection

We investigated the role of the Fas ligand (FasL)/Fas death pathway on apoptosis and cytokine production by T cells in Trypanosoma cruzi infection. Anti‐FasL, but not anti‐TNF‐α or anti‐TRAIL, blocked activation‐induced cell death of CD8 T cells and increased secretion of IL‐10 and IL‐4 by CD4 T cells from T. cruzi‐infected mice. CD4 and CD8 T cells up‐regulated Fas/FasL expression during T. cruzi infection. However, Fas expression increased earlier in CD8 T cells, and a higher proportion of CD8 T cells was activated and expressed IFN‐γ compared with CD4 T cells. Injection of anti‐FasL in infected mice reduced parasitemia and CD8 T cell apoptosis and increased the ratio of CD8:CD4 T cells recovered from spleen and peritoneum. FasL blockade increased the number of activated T cells, enhanced NO production, and reduced parasite loads in peritoneal macrophages. Injection of anti‐FasL increased IFN‐γ secretion by splenocytes responding to T. cruzi antigens but also exacerbated production of type 2 cytokines IL‐10 and IL‐4 at a late stage of acute infection. These results indicate that the FasL/Fas death pathway regulates apoptosis and coordinated cytokine responses by type 1 CD8 and type 2 CD4 T cells in T. cruzi infection.

Caspase-8 Activity Prevents Type 2 Cytokine Responses and Is Required for Protective T Cell-Mediated Immunity against Trypanosoma cruzi Infection

During Trypanosoma cruzi infection, T cells up-regulate caspase-8 activity. To assess the role of caspase-8 in T cell-mediated immunity, we investigated the effects of caspase-8 inhibition on T cells in viral FLIP (v-FLIP) transgenic mice. Compared with wild-type controls, increased parasitemia was observed in v-FLIP mice infected with T. cruzi. There was a profound decrease in expansion of both CD4 and CD8 T cell subsets in the spleens of infected v-FLIP mice. We did not find differences in activation ratios of T cells from transgenic or wild-type infected mice. However, the numbers of memory/activated CD4 and CD8 T cells were markedly reduced in v-FLIP mice, possibly due to defective survival. We also found decreased production of IL-2 and increased secretion of type 2 cytokines, IL-4 and IL-10, which could enhance susceptibility to infection. Similar, but less pronounced, alterations were observed in mice treated with the caspase-8 inhibitor, zIETD. Furthermore, blockade of caspase-8 by zIETD in vitro mimicked the effects observed on T. cruzi infection in vivo, affecting the generation of activated/memory T cells and T cell cytokine production. Caspase-8 is also required for NF-κB signaling upon T cell activation. Blockade of caspase-8 by either v-FLIP expression or treatment with zIETD peptide decreased NF-κB responses to TCR:CD3 engagement in T cell cultures. These results suggest a critical role for caspase-8 in the establishment of T cell memory, cell signaling, and regulation of cytokine responses during protozoan infection.

Myeloid-derived suppressor cells help protective immunity to Leishmania major infection despite suppressed T cell responses

Th1/Th2 cytokines play a key role in immune responses to Leishmania major by controlling macrophage activation for NO production and parasite killing. MDSCs, including myeloid precursors and immature monocytes, produce NO and suppress T cell responses in tumor immunity. We hypothesized that NO‐producing MDSCs could help immunity to L. major infection. Gr1hi(Ly6Chi) CD11bhi MDSCs elicited by L. major infection suppressed polyclonal and antigen‐specific T cell proliferation. Moreover, L. major‐induced MDSCs killed intracellular parasites in a NO‐dependent manner and reduced parasite burden in vivo. By contrast, treatment with ATRA, which induces MDSCs to differentiate into macrophages, increased development of lesions, parasite load, and T cell proliferation in draining LNs. Altogether, these results indicate that NO‐producing MDSCs help protective immunity to L. major infection, despite suppressed T cell proliferation.

Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection

In experimental Chagas’ disease, lymphocytes from mice infected with Trypanosoma cruzi show increased apoptosis in vivo and in vitro. Treatment with a pan‐caspase blocker peptide inhibited expression of the active form of effector caspase‐3 in vitro and rescued both B and T cells from cell death. Injection of the caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp(OMe)‐fluoromethyl ketone, but not a control peptide, reduced parasitemia and lymphocyte apoptosis in T. cruzi‐infected mice. Moreover, treatment with caspase inhibitor throughout acute infection increased the absolute numbers of B and T cells in the spleen and lymph nodes, without affecting cell infiltrates in the heart. Following treatment, we found increased accumulation of memory/activated CD4 and CD8 T cells, and secretion of IFN‐γ by splenocytes stimulated with T. cruzi antigens. Caspase inhibition in the course of infection reduced the intracellular load of parasites in peritoneal macrophages, and increased the production of TNF‐α and nitric oxide upon activation in vitro. Our results indicate that inhibition of caspases with a pan‐caspase blocker peptide improves protective type‐1 immune responses to T. cruzi infection. We suggest that mechanisms of apoptosis are potential therapeutic targets in Chagas’ disease.

Apoptosis differentially regulates mesenteric and subcutaneous lymph node immune responses to Trypanosoma cruzi

Infection with Trypanosoma cruzi causes expansion of subcutaneous (SLN) and atrophy of mesenteric (MLN) lymph nodes. Here we show that excision of MLN increased parasitemia in T. cruzi‐infected mice. We then studied how apoptosis of MLN cells affects immune responses to infection. T cell apoptosis increased in the MLN compared to SLN in T. cruzi‐infected mice. Absolute numbers of naïve T cells decreased, and activated T cells failed to accumulate in MLN during infection. In addition, activated T cells from MLN produced less IL‐2, IFN‐γ, IL‐4, and IL‐10 than T cells from SLN. Treatment with IL‐4 or with caspase‐9 inhibitor increased the recovery of viable T cells in vitro. Treatment with caspase‐9 inhibitor also increased the production of cytokines by MLN T cells from infected mice. Moreover, injection of a pan caspase inhibitor prevented MLN atrophy during T. cruzi infection. Caspase‐9, but not caspase‐8, inhibitor also reduced MLN atrophy and increased the recovery of naïve and activated T cells from MLN. These findings indicate that caspase‐mediated apoptosis and defective cytokine production are implicated in MLN atrophy and affect immune responses to T. cruzi infection.

Targeting caspases in intracellular protozoan infections

Caspases are cysteine aspartases acting either as initiators (caspases 8, 9, and 10) or executioners (caspases 3, 6, and 7) to induce programmed cell death by apoptosis. Parasite infections by certain intracellular protozoans increase host cell life span by targeting caspase activation. Conversely, caspase activation, followed by apoptosis of lymphocytes and other cells, prevents effective immune responses to chronic parasite infection. Here we discuss how pharmacological inhibition of caspases might affect the immunity to protozoan infections, by either blocking or delaying apoptosis.

Apoptotic CD8 T-lymphocytes disable macrophage-mediated immunity to Trypanosoma cruzi infection

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi. CD8 T-lymphocytes help to control infection, but apoptosis of CD8 T cells disrupts immunity and efferocytosis can enhance parasite infection within macrophages. Here, we investigate how apoptosis of activated CD8 T cells affects M1 and M2 macrophage phenotypes. First, we found that CD8 T-lymphocytes and inflammatory monocytes/macrophages infiltrate peritoneum during acute T. cruzi infection. We show that treatment with anti-Fas ligand (FasL) prevents lymphocyte apoptosis, upregulates type-1 responses to parasite antigens, and reduces infection in macrophages cocultured with activated CD8 T cells. Anti-FasL skews mixed M1/M2 macrophage profiles into polarized M1 phenotype, both in vitro and following injection in infected mice. Moreover, inhibition of T-cell apoptosis induces a broad reprogramming of cytokine responses and improves macrophage-mediated immunity to T. cruzi. The results indicate that disposal of apoptotic CD8 T cells increases M2-macrophage differentiation and contributes to parasite persistence.

Inhibition of caspase-8 activity reduces IFN-gamma expression by T cells from Leishmania major infection

Following infection with Leishmania major, T cell activation and apoptosis can be detected in draining lymph nodes of C57BL/6-infected mice. We investigated the mechanisms involved in apoptosis and cytokine expression following T cell activation. After two weeks of infection, apoptotic T cells were not detected in draining lymph nodes but activation with anti-CD3 induced apoptosis in both CD4 and CD8 T cells. Treatment with anti-Fas Ligand, caspase-8 or caspase- 9 inhibitors did not block activation-induced T-cell death. We also investigated whether the blockade of caspase-8 activity would affect the expression of type-1 or type-2 cytokines. At early stages of infection, both CD4 and CD8 T cells expressed IFN-gamma upon activation. Treatment with the caspase-8 inhibitor zIETD-fmk (benzyl-oxycarbonyl-Ile- Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone) reduced the proportion of CD8 T cells and IFN-gamma expression in both CD4 and CD8 T cells. We conclude that a non apoptotic role of caspase-8 activity may be required for T cell-mediated type-1 responses during L. major infection.

Inhibition of caspase-8 activity promotes protective Th1- and Th2-mediated immunity to Leishmania major infection

We investigated how apoptosis pathways mediated by death receptors and caspase‐8 affect cytokine responses and immunity to Leishmania major parasites. Splenic CD4 T cells undergo activation‐induced apoptosis, and blockade of FasL‐Fas interaction increased IFN‐γ and IL‐4 cytokine responses to L. major antigens. To block death receptor‐induced death, we used mice expressing a T cell‐restricted transgene for vFLIP. Inhibition of caspase‐8 activation in vFLIP mice enhanced Th1 and Th2 cytokine responses to L. major infection, even in the Th1‐prone B6 background. We also observed increased NO production by splenocytes from vFLIP mice upon T cell activation. Despite an exacerbated Th2 response, vFLIP mice controlled better L. major infection, with reduced lesions and lower parasite loads compared with WT mice. Moreover, injection of anti‐IL‐4 mAb in infected vFLIP mice disrupted control of parasite infection. Therefore, blockade of caspase‐8 activity in T cells improves immunity to L. major infection by promoting increased Th1 and Th2 responses.