Critical role of IL-25-ILC2-IL-5 axis in the production of anti-Francisella LPS IgM by B1 B cells.

B1 cells, a subset of B lymphocytes whose developmental origin, phenotype, and function differ from that of conventional B2 cells, are the main source of "natural" IgM but can also respond to infection by rapidly producing pathogen-specific IgM directed against T-independent antigens. Francisella tularensis (Ft) is a Gram-negative bacterium that causes tularemia. Infection with Ft Live Vaccine Strain activates B1 cells for production of IgM directed against the bacterial LPS in a process incompletely understood. Here we show that immunization with purified Ft LPS elicits production of LPS-specific IgM and IgG3 by B1 cells independently of TLR2 or MyD88. Immunization, but not infection, generated peritoneum-resident memory B1 cells that differentiated into LPS-specific antibody secreting cells (ASC) upon secondary challenge. IL-5 was rapidly induced by immunization with Ft LPS and was required for production of LPS-specific IgM. Antibody-mediated depletion of ILC2 indicated that these cells were the source of IL-5 and were required for IgM production. IL-25, an alarmin that strongly activates ILC2, was rapidly secreted in response to immunization or infection and its administration to mice significantly increased IgM production and B1 cell differentiation to ASC. Conversely, mice lacking IL-17RB, the IL-25 receptor, showed impaired IL-5 induction, IgM production, and B1 ASC differentiation in response to immunization. Administration of IL-5 to Il17rb-/- mice rescued these B1 cells-mediated responses. Il17rb-/- mice were more susceptible to infection with Ft LVS and failed to develop immunity upon secondary challenge suggesting that LPS-specific IgM is one of the protective adaptive immune mechanisms against tularemia. Our results indicated that immunization with Ft LPS triggers production of IL-25 that, through stimulation of IL-5 release by ILC2, promotes B1 cells activation and differentiation into IgM secreting cells. By revealing the existence of an IL-25-ILC2-IL-5 axis our results suggest novel strategies to improve vaccination against T-independent bacterial antigens.

A Novel Calcium-Dependent Protein Kinase 1 Inhibitor Potently Prevents Toxoplasma gondii Transmission to Foetuses in Mouse.

Treatments currently used to prevent congenital toxoplasmosis are non-specific of Toxoplasma gondii and have grievous side effects. To develop a more specific and less toxic drug, we have designed SP230, an imidazo[1,2-b]pyridazine salt targeting the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) and active against acute toxoplasmosis in mice. Efficiency of SP230 to inhibit foetal transmission of the parasite was evaluated in a mouse model of congenital toxoplasmosis. Swiss mice were infected at mid-pregnancy with tachyzoites or cysts of the ME49 strain of T. gondii by intraperitoneal and oral route, respectively, and treated with SP230 at 50 mg/kg for 5 days by the same routes. Parasite burden in organs of dams and in foetuses was measured by quantitative PCR. Intraperitoneal administration of SP230 drastically reduced the number of parasites (more than 97% of reduction) in the brain and lungs of dams, and led to a reduction of 66% of parasite burden in foetuses. Oral administration of SP230 was particularly efficient with 97% of reduction of parasite burdens in foetuses. SP230 did not impact number and weight of offspring in our conditions. This inhibitor of TgCDPK1 is a promising candidate for the development of alternative therapeutics to treat infected pregnant women.

Caspase-11-dependent pyroptosis of lung epithelial cells protects from melioidosis while caspase-1 mediates macrophage pyroptosis and production of IL-18.

Infection with Burkholderia pseudomallei or B. thailandensis triggers activation of the NLRP3 and NLRC4 inflammasomes leading to release of IL-1ß and IL-18 and death of infected macrophages by pyroptosis, respectively. The non-canonical inflammasome composed of caspase-11 is also activated by these bacteria and provides protection through induction of pyroptosis. The recent generation of bona fide caspase-1-deficient mice allowed us to reexamine in a mouse model of pneumonic melioidosis the role of caspase-1 independently of caspase-11 (that was also absent in previously generated Casp1-/- mice). Mice lacking either caspase-1 or caspase-11 were significantly more susceptible than wild type mice to intranasal infection with B. thailandensis. Absence of caspase-1 completely abolished production of IL-1ß and IL-18 as well as pyroptosis of infected macrophages. In contrast, in mice lacking caspase-11 IL-1ß and IL-18 were produced at normal level and macrophages pyroptosis was only marginally affected. Adoptive transfer of bone marrow indicated that caspase-11 exerted its protective action both in myeloid cells and in radio-resistant cell types. B. thailandensis was shown to readily infect mouse lung epithelial cells triggering pyroptosis in a caspase-11-dependent way in vitro and in vivo. Importantly, we show that lung epithelial cells do not express inflammasomes components or caspase-1 suggesting that this cell type relies exclusively on caspase-11 for undergoing cell death in response to bacterial infection. Finally, we show that IL-18's protective action in melioidosis was completely dependent on its ability to induce IFNγ production. In turn, protection conferred by IFNγ against melioidosis was dependent on generation of ROS through the NADPH oxidase but independent of induction of caspase-11. Altogether, our results identify two non-redundant protective roles for caspase-1 and caspase-11 in melioidosis: Caspase-1 primarily controls pyroptosis of infected macrophages and production of IL-18. In contrast, caspase-11 mediates pyroptosis of infected lung epithelial cells.

Production of anti-LPS IgM by B1a B cells depends on IL-1ß and is protective against lung infection with Francisella tularensis LVS.

The role of IL-1ß and IL-18 during lung infection with the gram-negative bacterium Francisella tularensis LVS has not been characterized in detail. Here, using a mouse model of pneumonic tularemia, we show that both cytokines are protective, but through different mechanisms. Il-18-/- mice quickly succumb to the infection and showed higher bacterial burden in organs and lower level of IFNγ in BALF and serum compared to wild type C57BL/6J mice. Administration of IFNγ rescued the survival of Il-18-/- mice, suggesting that their decreased resistance to tularemia is due to inability to produce IFNγ. In contrast, mice lacking IL-1 receptor or IL-1ß, but not IL-1α, appeared to control the infection in its early stages, but eventually succumbed. IFNγ administration had no effect on Il-1r1-/- mice survival. Rather, Il-1r1-/- mice were found to have significantly reduced titer of Ft LPS-specific IgM. The anti-Ft LPS IgM was generated in a IL-1ß-, TLR2-, and ASC-dependent fashion, promoted bacteria agglutination and phagocytosis, and was protective in passive immunization experiments. B1a B cells produced the anti-Ft LPS IgM and these cells were significantly decreased in the spleen and peritoneal cavity of infected Il-1b-/- mice, compared to C57BL/6J mice. Collectively, our results show that IL-1ß and IL-18 activate non-redundant protective responses against tularemia and identify an essential role for IL-1ß in the rapid generation of pathogen-specific IgM by B1a B cells.

Role of Canonical and Non-canonical Inflammasomes During Burkholderia Infection.

Burkholderia pseudomallei is a Gram-negative flagellate bacterium that causes melioidosis, a disease endemic to Southeast Asia and other tropical regions. Following infection of macrophages and other non-phagocytic cell types, B. pseudomallei or B. thailandensis (a related species that causes disease in mice but not humans) are able to escape the phagosome and replicate in the host cell cytoplasm. Resistance to infection with Burkholderia is dependent on the Nlrp3 and Nlrc4 inflammasomes and the non-canonical caspase-11 inflammasome. Nlrc4 mediates protection through induction of pyroptosis in the early phase of infection. As the infection progresses and as IL-18-dependent IFNγ production increases, caspase-11-dependent pyroptosis acquires a preponderant protective role. Production of IL-1ß and IL-18 during infection is primarily mediated by Nlrp3. IL-18 is essential for survival because of its ability to induce IFNγ production, which in turn activates macrophage microbicidal functions and primes for caspase-11 expression. In contrast, during melioidosis, IL-1ß has deleterious effects due to excessive recruitment of neutrophils to the lung and consequent tissue damage.

Intestinal CD103+ dendritic cells are key players in the innate immune control of Cryptosporidium parvum infection in neonatal mice.

Cryptosporidium parvum is a zoonotic protozoan parasite found worldwide, that develops only in the gastrointestinal epithelium and causes profuse diarrhea. Using a mouse model of C. parvum infection, we demonstrated by conditional depletion of CD11c+ cells that these cells are essential for the control of the infection both in neonates and adults. Neonates are highly susceptible to C. parvum but the infection is self-limited, whereas adults are resistant unless immunocompromised. We investigated the contribution of DC to the age-dependent susceptibility to infection. We found that neonates presented a marked deficit in intestinal CD103+ DC during the first weeks of life, before weaning, due to weak production of chemokines by neonatal intestinal epithelial cells (IEC). Increasing the number of intestinal CD103+ DC in neonates by administering FLT3-L significantly reduced susceptibility to the infection. During infections in neonates, the clearance of the parasite was preceded by a rapid recruitment of CD103+ DC mediated by CXCR3-binding chemokines produced by IEC in response to IFNγ. In addition to this key role in CD103+ DC recruitment, IFNγ is known to inhibit intracellular parasite development. We demonstrated that during neonatal infection CD103+ DC produce IL-12 and IFNγ in the lamina propria and the draining lymph nodes. Thus, CD103+DC are key players in the innate immune control of C. parvum infection in the intestinal epithelium. The relative paucity of CD103+ DC in the neonatal intestine contributes to the high susceptibility to intestinal infection.

Poly(I:C)-induced protection of neonatal mice against intestinal Cryptosporidium parvum infection requires an additional TLR5 signal provided by the gut flora.

The neonatal intestinal immune system is still undergoing development at birth, leading to a higher susceptibility to mucosal infections. In this study, we investigated the effect of poly(I:C) on controlling enteric infection by the protozoan Cryptosporidium parvum in neonatal mice. After poly(I:C) administration, a rapid reduction in parasite burden was observed and proved to be dependent on CD11c(+) cells and TLR3/TRIF signaling. Protection against C. parvum required additional signals provided by the gut flora through TLR5 and MyD88 signaling. This cooperation gave rise to higher levels of expression of critical mutually dependent cytokines such as interleukin 12p40 and type 1 and type 2 interferons, the last 2 being known to play a key role in the elimination of infected enterocytes. Our findings demonstrate in neonatal mice how gut flora synergizes with poly(I:C) to elicit protective intestinal immunity against an intracellular pathogen.

Specific Cell Targeting by Toxoplasma gondii Displaying Functional Single-Chain Variable Fragment as a Novel Strategy; A Proof of Principle.

Toxoplasma gondii holds significant therapeutic potential; however, its nonspecific invasiveness results in off-target effects. The purpose of this study is to evaluate whether T. gondii specificity can be improved by surface display of scFv directed against dendritic cells' endocytic receptor, DEC205, and immune checkpoint PD-L1. Anti-DEC205 scFv was anchored to the T. gondii surface either directly via glycosylphosphatidylinositol (GPI) or by fusion with the SAG1 protein. Both constructs were successfully expressed, but the binding results suggested that the anti-DEC-SAG1 scFv had more reliable functionality towards recombinant DEC protein and DEC205-expressing MutuDC cells. Two anti-PD-L1 scFv constructs were developed that differed in the localization of the HA tag. Both constructs were adequately expressed, but the localization of the HA tag determined the functionality by binding to PD-L1 protein. Co-incubation of T. gondii displaying anti-PD-L1 scFv with tumor cells expressing/displaying different levels of PD-L1 showed strong binding depending on the level of available biomarker. Neutralization assays confirmed that binding was due to the specific interaction between anti-PD-L1 scFv and its ligand. A mixed-cell assay showed that T. gondii expressing anti-PD-L1 scFv predominately targets the PD-L1-positive cells, with negligible off-target binding. The recombinant RH-PD-L1-C strain showed increased killing ability on PD-L1+ tumor cell lines compared to the parental strain. Moreover, a co-culture assay of target tumor cells and effector CD8+ T cells showed that our model could inhibit PD1/PD-L1 interaction and potentiate T-cell immune response. These findings highlight surface display of antibody fragments as a promising strategy of targeting replicative T. gondii strains while minimizing nonspecific binding.

Nasal administration of recombinant Neospora caninum secreting IL-15/IL-15Rα inhibits metastatic melanoma development in lung.

BACKGROUND: Metastases are the leading cause of mortality in many cancer types and lungs are one of the most common sites of metastasis alongside the liver, brain, and bones. In melanoma, 85% of late-stage patients harbor lung metastases. A local administration could enhance the targeting of metastases while limiting the systemic cytotoxicity. Therefore, intranasal administration of immunotherapeutic agents seems to be a promising approach to preferentially target lung metastases and decrease their burden on cancer mortality. From observations that certain microorganisms induce an acute infection of the tumor microenvironment leading to a local reactivating immune response, microbial-mediated immunotherapy is a next-generation field of investigation in which immunotherapies are engineered to overcome immune surveillance and escape from microenvironmental cancer defenses. METHODS: The goal of our study is to evaluate the potential of the intranasal administration of Neospora caninum in a syngeneic C57BL6 mouse model of B16F10 melanoma lung metastases. It also compares the antitumoral properties of a wild-type N. caninum versus N. caninum secreting human interleukin (IL)-15 fused to the sushi domain of the IL-15 receptor α chain, a potent activator of cellular immune responses. RESULTS: The treatment of murine lung metastases by intranasal administration of an N. caninum engineered to secrete human IL-15 impairs lung metastases from further progression with only 0,08% of lung surface harboring metastases versus 4,4% in wild-type N. caninum treated mice and 36% in untreated mice. The control of tumor development is associated with a strong increase in numbers, within the lung, of natural killer cells, CD8+ T cells and macrophages, up to twofold, fivefold and sixfold, respectively. Analysis of expression levels of CD86 and CD206 on macrophages surface revealed a polarization of these macrophages towards an antitumoral M1 phenotype. CONCLUSION: Administration of IL-15/IL-15Rα-secreting N. caninum through intranasal administration, a non-invasive route, lend further support to N. caninum-demonstrated clear potential as an effective and safe immunotherapeutic approach for the treatment of metastatic solid cancers, whose existing therapeutic options are scarce. Combination of this armed protozoa with an intranasal route could reinforce the existing therapeutic arsenal against cancer and narrow the spectrum of incurable cancers.

Neospora caninum: a new class of biopharmaceuticals in the therapeutic arsenal against cancer.

BACKGROUND: Microorganisms that can be used for their lytic activity against tumor cells as well as inducing or reactivating antitumor immune responses are a relevant part of the available immunotherapy strategies. Viruses, bacteria and even protozoa have been largely explored with success as effective human antitumor agents. To date, only one oncolytic virus-T-VEC-has been approved by the US Food and Drug Administration for use in biological cancer therapy in clinical trials. The goal of our study is to evaluate the potential of a livestock pathogen, the protozoan Neospora caninum, non-pathogenic in humans, as an effective and safe antitumorous agent. METHODS/RESULTS: We demonstrated that the treatment of murine thymoma EG7 by subcutaneous injection of N. caninum tachyzoites either in or remotely from the tumor strongly inhibits tumor development, and often causes their complete eradication. Analysis of immune responses showed that N. caninum had the ability to 1) lyze infected cancer cells, 2) reactivate the immunosuppressed immune cells and 3) activate the systemic immune system by generating a protective antitumor response dependent on natural killer cells, CD8-T cells and associated with a strong interferon (IFN)-γ secretion in the tumor microenvironment. Most importantly, we observed a total clearance of the injected agent in the treated animals: N. caninum exhibited strong anticancer effects without persisting in the organism of treated mice. We also established in vitro and an in vivo non-obese diabetic/severe combined immunodeficiency mouse model that N. caninum infected and induced a strong regression of human Merkel cell carcinoma. Finally, we engineered a N. caninum strain to secrete human interleukin (IL)-15, associated with the alpha-subunit of the IL-15 receptor thus strengthening the immuno-stimulatory properties of N. caninum. Indeed, this NC1-IL15hRec strain induced both proliferation of and IFN-γ secretion by human peripheral blood mononuclear cells, as well as improved efficacy in vivo in the EG7 tumor model. CONCLUSION: These results highlight N. caninum as a potential, extremely effective and non-toxic anticancer agent, capable of being engineered to either express at its surface or to secrete biodrugs. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine.

Babesia divergens glycosylphosphatidylinositols modulate blood coagulation and induce Th2-biased cytokine profiles in antigen presenting cells.

Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs.

The gut flora is required for the control of intestinal infection by poly(I:C) administration in neonates.

We found that immunostimulation of the intestinal immune system of neonatal mice by poly(I:C) injection decreased intestinal infection by the parasite Cryptosporidium parvum. We showed that the presence of dendritic cells and the cooperation of mutually dependent cytokines, such as IL-12p40, and type I and type II IFNs, were involved in the mechanism of protection induced by poly(I:C). This protection is dependent not only on TLR3-TRIF signaling, but also on the activation of the TLR5-MyD88 pathway by gut microbiota. These results raise the possibility that flagellated intestinal commensal bacteria may, in the presence of natural or synthetic agonists of TLR3, provide synergy between the TRIF and MyD88 signaling pathways, thereby favoring the development of mucosal defenses. In this addendum, we summarize these recent findings and discuss their implications for neonatal infections and immunomodulatory strategies.