Pedro Ferreirinha

Mucosal and systemic T cell response in mice intragastrically infected with Neospora caninum tachyzoites

The murine model has been widely used to study the host immune response to Neospora caninum. However, in most studies, the intraperitoneal route was preferentially used to establish infection. Here, C57BL/6 mice were infected with N. caninum tachyzoites by the intragastric route, as it more closely resembles the natural route of infection through the gastrointestinal tract. The elicited T-cell mediated immune response was evaluated in the intestinal epithelium and mesenteric lymph nodes (MLN). Early upon the parasitic challenge, IL-12 production by conventional and plasmacytoid dendritic cells was increased in MLN. Accordingly, increased proportions and numbers of TCRαβ+CD8+IFN-γ+ lymphocytes were detected, not only in the intestinal epithelium and MLN, but also in the spleen of the infected mice. In this organ, IFN-γ-producing TCRαβ+CD4+ T cells were also found to increase in the infected mice, however later than CD8+ T cells. Interestingly, splenic and MLN CD4+CD25+ T cells sorted from infected mice presented a suppressive activity on in vitro T cell proliferation and cytokine production above that of control counterparts. These results altogether indicate that, by producing IFN-γ, TCRαβ+CD8+ cells contribute for local and systemic host protection in the earliest days upon infection established through the gastrointestinal tract. Nevertheless, they also provide substantial evidence for a parasite-driven reinforcement of T regulatory cell function which may contribute for parasite persistence in the host and might represent an additional barrier to overcome towards effective vaccination.

Immune response in the adipose tissue of lean mice infected with the protozoan parasite Neospora caninum

The adipose tissue can make important contributions to immune function. Nevertheless, only a limited number of reports have investigated in lean hosts the immune response elicited in this tissue upon infection. Previous studies suggested that the intracellular protozoan Neospora caninum might affect adipose tissue physiology. Therefore, we investigated in mice challenged with this protozoan if immune cell populations within adipose tissue of different anatomical locations could be differently affected. Early in infection, parasites were detected in the adipose tissue and by 7 days of infection increased numbers of macrophages, regulatory T (Treg) cells and T‐bet+ cells were observed in gonadal, mesenteric, omental and subcutaneous adipose tissue. Increased expression of interferon‐γ was also detected in gonadal adipose tissue of infected mice. Two months after infection, parasite DNA was no longer detected in these tissues, but T helper type 1 (Th1) cell numbers remained above control levels in the infected mice. Moreover, the Th1/Treg cell ratio was higher than that of controls in the mesenteric and subcutaneous adipose tissue. Interestingly, chronically infected mice presented a marked increase of serum leptin, a molecule that plays a role in energy balance regulation as well as in promoting Th1‐type immune responses. Altogether, we show that an apicomplexa parasitic infection influences immune cellular composition of adipose tissue throughout the body as well as adipokine production, still noticed at a chronic phase of infection when parasites were already cleared from that particular tissue. This strengthens the emerging view that infections can have long‐term consequences for the physiology of adipose tissue.

Predominant role of interferon-γ in the host protective effect of CD8 + T cells against Neospora caninum infection

It is well established that CD8+ T cells play an important role in protective immunity against protozoan infections. However, their role in the course of Neospora caninum infection has not been fully elucidated. Here we report that CD8-deficient mice infected with N. caninum presented higher parasitic loads in the brain and lungs and lower spleen and brain immunity-related GTPases than their wild-type counterparts. Moreover, adoptive transfer of splenic CD8+ T cells sorted from N. caninum-primed immunosufficient C57BL/10 ScSn mice prolonged the survival of infected IL-12-unresponsive C57BL/10 ScCr recipients. In both C57BL/6 and C57BL/10 ScSn mice CD8+ T cells are activated and produce interferon-γ (IFN-γ) upon challenged with N. caninum. The host protective role of IFN-γ produced by CD8+ T cells was confirmed in N. caninum-infected RAG2-deficient mice reconstituted with CD8+ T cells obtained from either IFN-γ-deficient or wild-type donors. Mice receiving IFN-γ-expressing CD8+ T cells presented lower parasitic burdens than counterparts having IFN-γ-deficient CD8+ T cells. Moreover, we observed that N. caninum-infected perforin-deficient mice presented parasitic burdens similar to those of infected wild-type controls. Altogether these results demonstrate that production of IFN-γ is a predominant protective mechanism conferred by CD8+ T cells in the course of neosporosis.

Enrichment of IFN-γ producing cells in different murine adipose tissue depots upon infection with an apicomplexan parasite

Here we report that lean mice infected with the intracellular parasite Neospora caninum show a fast but sustained increase in the frequency of IFN-γ-producing cells noticeable in distinct adipose tissue depots. Moreover, IFN-γ-mediated immune memory could be evoked in vitro in parasite antigen-stimulated adipose tissue stromal vascular fraction cells collected from mice infected one year before. Innate or innate-like cells such as NK, NK T and TCRγδ+ cells, but also CD4+ and CD8+ TCRβ+ lymphocytes contributed to the IFN-γ production observed since day one of infection. This early cytokine production was largely abrogated in IL-12/IL23 p40-deficient mice. Moreover, production of IFN-γ by stromal vascular fraction cells isolated from these mice was markedly lower than that of wild-type counterparts upon stimulation with parasite antigen. In wild-type mice the increased IFN-γ production was concomitant with up-regulated expression of genes encoding interferon-inducible GTPases and nitric oxide synthase, which are important effector molecules in controlling intracellular parasite growth. This increased gene expression was markedly impaired in the p40-deficient mice. Overall, these results show that NK cells but also diverse T cell populations mediate a prompt and widespread production of IFN-γ in the adipose tissue of N. caninum infected mice.

Protective effect of intranasal immunization with Neospora caninum membrane antigens against murine neosporosis established through the gastrointestinal tract

Neospora caninum is an Apicomplexa parasite that in the last two decades was acknowledged as the main pathogenic agent responsible for economic losses in the cattle industry. In the present study, the effectiveness of intranasal immunization with N. caninum membrane antigens plus CpG adjuvant was assessed in a murine model of intragastrically established neosporosis. Immunized mice presented a lower parasitic burden in the brain on infection with 5 × 107 tachyzoites, showing that significant protection was achieved by this immunization strategy. Intestinal IgA antibodies raised by immunization markedly agglutinated live N. caninum tachyzoites whereas previous opsonization with IgG antibodies purified from immunized mice sera reduced parasite survival within macrophage cells. Although an IgG1 : IgG2a ratio < 1 was detected in the immunized mice before and after infection, indicative of a predominant T helper type 1 immune response, no increased production of interferon‐γ was detected in the spleen or mesenteric lymph nodes of the immunized mice. Altogether, these results show that mucosal immunization with N. caninum membrane proteins plus CpG adjuvant protect against intragastrically established neosporosis and indicate that parasite‐specific mucosal and circulating antibodies have a protective role against this parasitic infection.

Celecoxib promotes degranulation of CD8(+) T cells in HPV-induced lesions of K14-HPV16 transgenic mice.

AIMS Human papillomavirus (HPV) is a known biologic carcinogen which is commonly transmitted through sexual intercourse. CD8(+) T cells are known effectors against tumour cells and an important prognostic marker in HPV-induced cancers. COX-2 inhibitors enhance CD8(+) T cell activity against some cancers. In this work, we sought to study the presence and activation of CD8(+) T lymphocytes in lesions from K14-HPV16 transgenic mice and the immunomodulatory effect of celecoxib (CXB) over these cells. MAIN METHODS Skin samples of CXB-treated and untreated HPV16(-/-) and HPV16(+/-) mice were enzymatically digested and analysed by flow cytometry to assess CD8(+) and CD8(+)CD107a(+) T cell infiltrates. Matched skin samples were classified histologically. KEY FINDINGS HPV16(+/-) mice presented higher CD8(+) T cell infiltration than HPV16(-/-) animals (P<0.001). Older HPV16(+/-) animals showed epidermal dysplasia and increased percentages of CD8(+)CD107a(+) T cells compared with younger animals with hyperplasia (P<0.001), validating this model for testing the effects of celecoxib on CD8(+) T cells. CXB-treated HPV16(+/-) mice showed higher percentages of CD8(+)CD107a(+) T cells compared with untreated HPV16(+/-) animals (P<0.01), but no differences were observed concerning the progression of epidermal lesions. SIGNIFICANCE These findings indicate that celecoxib enhances the degranulation of CD8(+) T cells on HPV16-induced lesions, suggesting the potential clinical use of COX-2 inhibitors. Additionally, this study demonstrates the usefulness of the K14-HPV16 mouse model for testing therapeutic immunomodulatory approaches.

Mucosal immunization confers long-term protection against intragastrically established Neospora caninum infection.

Neospora caninum is an obligate intracellular protozoan parasite responsible for heavy economic losses in dairy and beef cattle farms worldwide. Although vaccination is widely regarded as the preferable strategy to prevent neosporosis no commercial vaccine is currently available. We have previously shown that intranasal immunization with an N. caninum antigen extract enriched in hydrophobic proteins plus CpG adjuvant protected mice against intragastrically established neosporosis. Nevertheless, the antigen specificity as well as the long-term protective effect of this immunization strategy were not determined. Here, we show that the protective effect of this intranasal immunization procedure lasted for at least 20weeks. Protection was accompanied by long-lasting elevated levels of parasite-specific serum IgG and intestinal IgA. Moreover, spleen and mesenteric lymph node cells obtained from non-infected long-term immunized mice responded by producing interferon-γ following in vitro parasite-antigen recall. Analysis of serum IgG and intestinal IgA antibody reactivity in immunized mice identified dense granule antigen 7 (NcGRA7) and microneme associated protein 1 (NcMIC1) as immunodominant antigens respectively recognized by those antibody fractions. In summary, this work shows that a previously reported mucosal immunization strategy against N. caninum infection established through the gastrointestinal tract is effective in the long term.

Poly-N-Acetylglucosamine Production by Staphylococcus epidermidis Cells Increases Their In Vivo Proinflammatory Effect.

ABSTRACT Poly-N-acetylglucosamine (PNAG) is a major component of the Staphylococcus epidermidis biofilm extracellular matrix. However, it is not yet clear how this polysaccharide impacts the host immune response and infection-associated pathology. Faster neutrophil recruitment and bacterial clearance were observed in mice challenged intraperitoneally with S. epidermidis biofilm cells of the PNAG-producing 9142 strain than in mice similarly challenged with the isogenic PNAG-defective M10 mutant. Moreover, intraperitoneal priming with 9142 cells exacerbated liver inflammatory pathology induced by a subsequent intravenous S. epidermidis challenge, compared to priming with M10 cells. The 9142-primed mice had elevated splenic CD4+ T cells producing gamma interferon and interleukin-17A, indicating that PNAG promoted cell-mediated immunity. Curiously, despite having more marked liver tissue pathology, 9142-primed mice also had splenic T regulatory cells with greater suppressive activity than those of their M10-primed counterparts. By showing that PNAG production by S. epidermidis biofilm cells exacerbates host inflammatory pathology, these results together suggest that this polysaccharide contributes to the clinical features associated with biofilm-derived infections.

Interferon-γ-dependent protection against Neospora caninum infection conferred by mucosal immunization in IL-12/IL-23 p40-deficient mice

We have recently demonstrated the effectiveness of an intranasal immunization approach against Neospora caninum infection in immunosufficient mice. Generated evidence indicated that antibodies could be mediating the observed protection. We similarly immunized IL-12/IL-23 p40 chain-deficient (Il12b-/-) mice, which have impaired cellular immunity, to further explore the host protective mechanism conferred by the used immunization strategy. The immunized mice presented lower parasitic burdens after intraperitoneal infection with N. caninum and also had elevated levels of parasite-specific antibodies. However, passive immunization with antibodies purified from immunized donors conferred only limited protection to infected Il12b-/- recipients. Despite their intrinsic IL-12 deficiency, the immunized Il12b-/- mice mounted a parasite-specific immune response that was mediated by interferon-γ (IFN-γ). Neutralization of IFN-γ in the immunized mice abrogated the observed protective effect of the immunization. These results show altogether that the used immunization strategy overcome the cellular immunity defect of Il12b-/- mice and conferred protection from N. caninum infection. The observed protective effect was predominantly mediated by IFN-γ and to a lesser extent but non-negligibly by IgG antibodies. These results also highlight that in a host with compromised cellular immunity, the immune response against intracellular pathogens could be markedly boosted by immunization.

Abstract 4047: NF-kB inhibitor DMAPT enhances cisplatin efficacy and reduces its toxicity in a carcinogen-induced model of muscle-invasive bladder cancer

BACKGROUND: Nuclear factor kappaB (NF-κB) blocks apoptosis and promotes chemoresistance to drugs like cisplatin (CDDP). However, the clinical use of NF-κB inhibitors is restricted to hematological malignancies and the benefit of NF-κB inhibition in solid tumors has yet to be realized. The novel agent dimethylaminoparthenolide (DMAPT) blocks NF-κB in bladder cancer cell lines and can overcome chemoresistance as part of a combination therapy. METHODS AND RESULTS: We tested DMAPT9s ability to enhance CDDP9s efficacy against muscle-invasive bladder cancer. In vitro, 2.5 µM DMAPT was shown to up-regulate death receptor 4 and 5 expression and induce poly (ADP-ribose) polymerase (PARP) cleavage in UMUC-3 cells. Both 2.5 µM DMAPT and 2.5 µM CDDP reduced UMUC-3 cell viability as single agents, and their effect was enhanced when combined. We next tested CDDP (4.0 mg/kg/week i.p) and DMAPT (100.0 mg/kg/day oral) in combination and as single agents for 6 weeks in an immune-competent mouse model of muscle-invasive (T2) bladder cancer induced by the tobacco-related agent N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Procedures followed the 2010/63/EU animal welfare directive. One DMAPT-treated and 1 CDDP-treated mouse died during the experiments. The cohorts were: negative control (no BBN exposure, n=15), positive control (BBN but no therapy, n=19), BBN and DMAPT-treated (n=17), BBN and CDDP-treated (n=18) and BBN and the combination of CDDP plus DMAPT (n=19). Negative control, positive control, DMAPT, CDDP and combination-treated mice showed 0.0%, 42.1%, 35.3%, 11.1% and 0.0% bladder cancer incidence, respectively. T2 (muscle-invasive) lesions were present in positive controls (15.8%), DMAPT (17.6%) and CDDP (5.6%), but not in the combination group (0.0%). High-grade urothelial dysplasia, a pre-malignant lesion, was observed at rates of: 57.9% (positive controls), 29.4% (DMAPT), 16.6% (CDDP) and 0.0% (combination). CDDP induced renal tubular necrosis and interstitial nephritis seen on HE 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4047. doi:10.1158/1538-7445.AM2017-4047