Characterization of an African Swine Fever Virus Field Isolate from Vietnam with Deletions in the Left Variable Multigene Family Region.

In this paper, we report the characterization of a genetically modified live-attenuated African swine fever virus (ASFV) field strain isolated from Vietnam. The isolate, ASFV-GUS-Vietnam, belongs to p72 genotype II, has six multi-gene family (MGF) genes deleted, and an Escherichia coli GusA gene (GUS) inserted. When six 6-8-week-old pigs were inoculated with ASFV-GUS-Vietnam oro-nasally (2 × 105 TCID50/pig), they developed viremia, mild fever, lethargy, and inappetence, and shed the virus in their oral and nasal secretions and feces. One of the pigs developed severe clinical signs and was euthanized 12 days post-infection, while the remaining five pigs recovered. When ASFV-GUS-Vietnam was inoculated intramuscularly (2 × 103 TCID50/pig) into four 6-8 weeks old pigs, they also developed viremia, mild fever, lethargy, inappetence, and shed the virus in their oral and nasal secretions and feces. Two contact pigs housed together with the four intramuscularly inoculated pigs, started to develop fever, viremia, loss of appetite, and lethargy 12 days post-contact, confirming horizontal transmission of ASFV-GUS-Vietnam. One of the contact pigs died of ASF on day 23 post-contact, while the other one recovered. The pigs that survived the exposure to ASFV-GUS-Vietnam via the mucosal or parenteral route were fully protected against the highly virulent ASFV Georgia 2007/1 challenge. This study showed that ASFV-GUS-Vietnam field isolate is able to induce complete protection in the majority of the pigs against highly virulent homologous ASFV challenge, but has the potential for horizontal transmission, and can be fatal in some animals. This study highlights the need for proper monitoring and surveillance when ASFV live-attenuated virus-based vaccines are used in the field for ASF control in endemic countries.

Crosspteryx fibrifuga leaf extract enhances host resistance to Trypanosoma congolense infection in mice by regulating host immune response and disrupting the activity of parasite superoxide dismutase enzyme.

African trypanosomiasis, a neglected tropical disease, is caused by diverse species of the protozoan parasite belonging to the genus Trypanosoma. Although anti-trypanosomal medications exist, the increase in drug resistance and persistent antigenic variation has necessitated the development of newer and more efficacious therapeutic agents which are selectively toxic to the parasite. In this study, we assessed the trypanocidal efficacy of Crosspteryx fibrifuga leaf extract (C.f/L-extract) in vitro. Following treatment of T. congolense parasites with C.f/L-extract, we observed a significant decrease in parasite number and an elevation in the expression of the apoptotic markers, Annexin V and 7-Aminoactinomycin D (7AAD). Interestingly, at the same concentration (50 µg/mL), C.f/L-extract was not cytotoxic to murine whole splenocytes. We also observed a significant increase in pro-inflammatory cytokines and nitric oxide secretion by bone marrow derived macrophages following treatment with C.f/L-extract (10 µg/mL and 50 µg/mL) compared to PBS treated controls, suggesting that the extract possesses an immune regulatory effect. Treatment of T. congolense infected mice with C.f/L-extract led to significant decrease in parasite numbers and a modest increase in mouse survival compared to PBS treated controls. In addition, there was a significant increase in CD4+IFN-γ+ T cells and a decrease in CD4+IL-10+ T cells in the spleens of T. congolense infected mice treated with C.f/L-extract. Interestingly, C.f/L-extract treatment decreased the activity of superoxide dismutase (an enzyme that protects unicellular organisms from oxidative stress) in T. congolense parasites but not in splenocytes. Collectively, our study has identified C.f/L-extract as a potential anti-trypanosomal agent that warrant further investigation and possibly explored as a treatment option for T. congolense infection.

Evaluation of a Lateral Flow Assay for Rapid Detection of African Swine Fever Virus in Multiple Sample Types.

Antibody-based lateral flow assay (LFA) is a quick and inexpensive tool used to detect pathogens in field samples, especially in hard-to-reach remote areas that may have limited access to central laboratories during an outbreak or surveillance. In this study, we investigated the ability of a commercially available LFA, PenCheck®, to detect African swine fever virus (ASFV) in clinical samples derived from pigs infected with highly virulent ASFV strains. The assay was specific and positively identified the majority of pigs showing high fever during the early stages (between 3 and 5 days) of infection. PenCheck® LFA also detected ASFV in serum and tissue samples collected from pigs that succumbed to experimental ASFV infection and whole blood, plasma, and tissue samples from the field. The limit of detection of the assay was ASFV titer 107.80 TCID50/mL, corresponding to ASFV real-time PCR values below 23 Ct. Although the sensitivity of the assay is less than that of the laboratory-based real-time PCR assays, the results obtained with the PenCheck® LFA in this study suggest that it can be used as a herd-level, field-deployable, and easy-to-use diagnostic tool to identify ASF-affected farms when access to portable molecular assays or central laboratories is not possible.

Superficial Inguinal Lymph Nodes for Screening Dead Pigs for African Swine Fever.

African swine fever (ASF) has spread across the globe and has reached closer to North America since being reported in the Dominican Republic and Haiti. As a result, surveillance measures have been heightened and the utility of alternative samples for herd-level monitoring and dead pig sampling have been investigated. Passive surveillance based on the investigation of dead pigs, both domestic and wild, plays a pivotal role in the early detection of an ASF incursion. The World Organization for Animal Health (OIE)-recommended samples for dead pigs are spleen, lymph nodes, bone marrow, lung, tonsil and kidney. However, obtaining these samples requires opening up the carcasses, which is time-consuming, requires skilled labour and often leads to contamination of the premises. As a result, we investigated the suitability of superficial inguinal lymph nodes (SILNs) for surveillance of dead animals. SILNs can be collected in minutes with no to minimum environmental contamination. Here, we demonstrate that the ASF virus (ASFV) genome copy numbers in SILNs highly correlate with those in the spleen and, by sampling SILN, we can detect all pigs that succumb to highly virulent and moderately virulent ASFV strains (100% sensitivity). ASFV was isolated from all positive SILN samples. Thus, sampling SILNs could be useful for routine surveillance of dead pigs on commercial and backyard farms, holding pens and dead on arrival at slaughter houses, as well as during massive die-offs of pigs due to unknown causes.

Meat Exudate for Detection of African Swine Fever Virus Genomic Material and Anti-ASFV Antibodies.

African swine fever (ASF) is one of the most important viral diseases of pigs caused by the ASF virus (ASFV). The virus is highly stable over a wide range of temperatures and pH and can survive in meat and meat products for several months, leading to long-distance transmission of ASF. Whole blood, serum, and organs from infected pigs are used routinely as approved sample types in the laboratory diagnosis of ASF. However, these sample types may not always be available. Here, we investigated meat exudate as an alternative sample type for the detection of ASFV-specific nucleic acids and antibodies. Pigs were infected with various ASFV strains: the highly virulent ASFV Malawi LIL 18/2 strain, the moderately-virulent ASFV Estonia 2014 strain, or the low-virulent ASFV OURT/88/3 strain. The animals were euthanized on different days post-infection (dpi), and meat exudates were collected and tested for the presence of ASFV-specific nucleic acids and antibodies. Animals infected with the ASFV Malawi LIL 18/2 developed severe clinical signs and succumbed to the infection within seven dpi, while pigs infected with ASFV Estonia 2014 also developed clinical signs but survived longer, with a few animals seroconverting before succumbing to the ASFV infection or being euthanized as they reached humane endpoints. Pigs infected with ASFV OURT/88/3 developed transient fever and seroconverted without mortality. ASFV genomic material was detected in meat exudate from pigs infected with ASFV Malawi LIL 18/2 and ASFV Estonia 2014 at the onset of viremia but at a lower amount when compared to the corresponding whole blood samples. Low levels of ASFV genomic material were detected in the whole blood of ASFV OURT/88/3-infected pigs, and no ASFV genomic material was detected in the meat exudate of these animals. Anti-ASFV antibodies were detected in the serum and meat exudate derived from ASFV OURT/88/3-infected pigs and in some of the samples derived from the ASFV Estonia 2014-infected pigs. These results indicate that ASFV genomic material and anti-ASFV antibodies can be detected in meat exudate, indicating that this sample can be used as an alternative sample type for ASF surveillance when routine sample types are unavailable or are not easily accessible.

Critical Roles of Phosphoinositide 3-Kinase δ in the Humoral Immune Response to Trypanosoma congolense Infection.

PI3Kδ is critical in generating humoral and regulatory immune responses. In this study, we determined the impact of PI3Kδ in immunity to Trypanosoma congolense, an African trypanosome that can manipulate and evade Ab responses critical for protection. Upon infection with T. congolense, PI3KδD910A mice lacking PI3Kδ activity paradoxically show a transient enhancement in early control of parasitemia, associated with impaired production of regulatory IL-10 by B cells in the peritoneum. C57BL/6 wild-type (WT) mice treated with the PI3Kδ inhibitor (PI3Kδi) Idelalisib showed a similar transient decrease in parasitemia associated with reduced IL-10. Strikingly, however, we find that PI3KδD910A mice were ultimately unable to control this infection, resulting in uncontrolled parasitemia and death within 2 wk. Assessment of humoral responses revealed delayed B cell activation, impaired germinal center responses, and compromised Ab responses to differing degrees in PI3KδD910A and PI3Kδi-treated mice. To test the role of Abs, we administered serum from WT mice to PI3KδD910A mice and found that lethality was prevented by postinfection serum. Interestingly, serum from naive WT mice provided partial protection to PI3KδD910A mutants, indicating an additional role for natural Abs. Together our findings suggest that although PI3Kδ drives immune regulatory responses that antagonize early control of parasite growth in the peritoneum, it is also required for generation of Abs that are critical for protection from systemic trypanosome infection. The essential role of PI3Kδ for host survival of African trypanosome infection contrasts with findings for other pathogens such as Leishmania, underlining the critical importance of PI3Kδ-dependent humoral immunity in this disease.

Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5' untranslated region (5' UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

Rapid and highly sensitive portable detection of African swine fever virus.

African swine fever (ASF) continues to spread across Asia, devastating pig populations. The disease is nearly 100% fatal in pigs, and currently, there is no effective vaccine available. Therefore, early detection of ASF is critical for effective disease control. The testing process usually requires samples to be shipped to a central laboratory, which may take many hours of travel or shipping time, delaying the results needed for a rapid response. The ability to confirm ASFV-infected animals on-site or in a regional laboratory that has limited technical capacity and/or infrastructure should eliminate these issues. This study describes the successful transfer of a highly sensitive and specific laboratory-validated real-time PCR assay to a portable pen-side thermocycler, which can be operated in the field for rapid detection of ASFV following a quick manual nucleic acid extraction from a wide array of clinical samples including aggregate samples such as oral fluids. The performance of the portable assay was comparable to the laboratory-based assay. The true portability of the assay was evaluated in seven ASF-suspected farms in Vietnam by testing eighty-nine freshly collected whole blood samples on-site. The results obtained on-site were in agreement with the laboratory data obtained the following day. Availability of this field-deployable molecular assay would eliminate the need to ship samples to a central laboratory, when rapid laboratory results are required, ultimately improving the response time.

Leishmania Immunity: Advancing Immunotherapy and Vaccine Development.

Parasitic diseases still constitute a major global health problem affecting billions of people around the world. These diseases are capable of becoming chronic and result in high morbidity and mortality. Worldwide, millions of people die each year from parasitic diseases, with the bulk of those deaths resulting from parasitic protozoan infections. Leishmaniasis, which is a disease caused by over 20 species of the protozoan parasite belonging to the genus Leishmania, is an important neglected disease. According to the World Health Organization (WHO), an estimated 12 million people are currently infected in about 98 countries and about 2 million new cases occur yearly, resulting in about 50,000 deaths each year. Current treatment methods for leishmaniasis are not very effective and often have significant side effects. In this review, we discussed host immunity to leishmaniasis, various treatment options currently being utilized, and the progress of both immunotherapy and vaccine development strategies used so far in leishmaniasis. We concluded with insights into what the future holds toward the fight against this debilitating parasitic disease.

Identification of a Protective Leishmania Antigen Dihydrolipoyl Dehydrogenase and Its Responding CD4+ T Cells at Clonal Level.

There is currently no clinically effective vaccine against cutaneous leishmaniasis because of poor understanding of the Ags that elicit protective CD4+ T cell immunity. In this study, we identified a naturally processed peptide (DLD63-79) that is derived from Leishmania dihydrolipoyl dehydrogenase (DLD) protein. DLD is conserved in all pathogenic Leishmania species, is expressed by both the promastigote and amastigote stages of the parasite, and elicits strong CD4+ T cell responses in mice infected with L. major We generated I-Ab-DLD63-79 tetramer and identified DLD-specific CD4+ T cells at clonal level. Following L. major infection, DLD63-79-specific CD4+ T cells massively expanded and produced effector cytokines (IFN-γ and TNF). This was followed by a gradual contraction, stable maintenance following lesion resolution, and display of memory (recall) response following secondary challenge. Vaccination with rDLD protein induced strong protection in mice against virulent L. major challenge. Identification of Ags that elicit protective immunity and their responding Ag-specific T cells are critical steps necessary for developing effective vaccines and vaccination strategies against infectious agents, including protozoan parasites.

Immune System Effects of Insulin-Like Peptide 5 in a Mouse Model.

Introduction: Insulin-like peptide 5 (INSL5) is a peptide hormone with proposed actions in glucose homeostasis and appetite regulation via its cognate receptor, relaxin family peptide receptor 4 (RXFP4). Here, we look for evidence for their involvement in the immune system using a mouse model. Methods: In silico analyses: we queried public databases for evidence of expression of INSL5-RXFP4 in immune system tissues/cells (NCBI's SRA and GeoProfiles) and disorders (EMBO-EBI) and performed phylogenetic footprinting to look for evidence that they are regulated by immune-associated transcription factors (TFs). Experimental analyses: We characterized the expression and correlation of INSL5/RXFP4 and other immune system markers in central and peripheral immune organs from C57/bl6 mice in seven cohorts. We tested whether fluctuations in circulating INSL5 induce an immune response, by injecting mice with 30 µg/kg of INSL5 peptide in the peritoneum, and examining levels of immune markers and metabolic peptides in plasma. Lastly, we quantified the expression of Rxfp4 in T-cells, dendritic cells and cell lines derived from human and mouse and tested the hypothesis that co-incubation of ANA-1 cells in INSL5 and LPS alters cytokine expression. Results: We find Insl5 expression only in thymus (in addition to colon) where its expression was highly correlated with Il-7, a marker of thymocyte development. This result is consistent with our in silico findings that Insl5 is highly expressed in thymic DP, DN thymocytes and cortical TEC's, and with evidence that it is regulated by thymocyte-associated TF's. We find Rxfp4 expression in all immune organs, and moderately high levels in DCs, particularly splenic DCs, and evidence that it is regulated by immune-associated TF's, such as STAT's and GATA. Systemic effects: We observed significantly elevated concentrations of blood GLP-1, GIP, GCG and PYY following intraperitoneal injection of INSL5, and significantly altered expression of cytokines IL-5, IL-7, M-CSF, IL-15, IL-27 and MIP-2. Immune cell effects: Incubation of ANA-1 cells with INSL5 impeded cell growth and led to a transient elevation of IL-15 and sustained reduction in IL-1ß, IL-6 and TNFα. Conclusion: We propose that INSL5-RXFP4 play a novel role in both central and peripheral immune cell signaling.

Semaphorin 3E Regulates the Response of Macrophages to Lipopolysaccharide-Induced Systemic Inflammation.

Semaphorin 3E (Sema3E) is a secreted protein that was initially discovered as a neuronal guidance cue. Recent evidence showed that Sema3E plays an essential role in regulating the activities of various immune cells. However, the exact role of Sema3E in macrophage function, particularly during inflammation, is not fully understood. We studied the impact of Sema3E gene deletion on macrophage function during the LPS-induced acute inflammatory response. We found that Sema3E-deficient (Sema3e-/- ) mice were better protected from LPS-induced acute inflammation as exemplified by their superior clinical score and effective temperature control compared with their wild-type littermates. This superior control of inflammatory response in Sema3e-/- mice was associated with significantly lower phosphorylation of ERK1/2, AKT, STAT3, and NF-κB, and a concomitant reduction in inducible NO synthase expression and production of TNF and IL-6 compared with their Sema3e+/+ littermates. Sema3e-/- mice also contained significantly higher numbers of activated macrophages compared with their Sema3e+/+ littermates at both baselines and after LPS challenge. In vivo-specific deletion of the Sema3E high-affinity receptor, plexinD1, on macrophages led to the improvement in clinical disease following exposure to a lethal dose of LPS. Collectively, our data show that Sema3E plays an essential role in dampening the early inflammatory response to LPS by regulating macrophage function, suggesting an essential role of this pathway in macrophage inflammatory response.

TLR-2 and MyD88-Dependent Activation of MAPK and STAT Proteins Regulates Proinflammatory Cytokine Response and Immunity to Experimental Trypanosoma congolense Infection.

It is known that Trypanosoma congolense infection in mice is associated with increased production of proinflammatory cytokines by macrophages and monocytes. However, the intracellular signaling pathways leading to the production of these cytokines still remain unknown. In this paper, we have investigated the innate receptors and intracellular signaling pathways that are associated with T. congolense-induced proinflammatory cytokine production in macrophages. We show that the production of IL-6, IL-12, and TNF-α by macrophages in vitro and in vivo following interaction with T. congolense is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) including ERK, p38, JNK, and signal transducer and activation of transcription (STAT) proteins. Specific inhibition of MAPKs and STATs signaling pathways significantly inhibited T. congolense-induced production of proinflammatory cytokines in macrophages. We further show that T. congolense-induced proinflammatory cytokine production in macrophages is mediated via Toll-like receptor 2 (TLR2) and involves the adaptor molecule, MyD88. Deficiency of MyD88 and TLR2 leads to impaired cytokine production by macrophages in vitro and acute death of T. congolense-infected relatively resistant mice. Collectively, our results provide insight into T. congolense-induced activation of the immune system that leads to the production of proinflammatory cytokines and resistance to the infection.

Host Immune Responses and Immune Evasion Strategies in African Trypanosomiasis.

Parasites, including African trypanosomes, utilize several immune evasion strategies to ensure their survival and completion of their life cycles within their hosts. The defense factors activated by the host to resolve inflammation and restore homeostasis during active infection could be exploited and/or manipulated by the parasites in an attempt to ensure their survival and propagation. This often results in the parasites evading the host immune responses as well as the host sustaining some self-inflicted collateral tissue damage. During infection with African trypanosomes, both effector and suppressor cells are activated and the balance between these opposing arms of immunity determines susceptibility or resistance of infected host to the parasites. Immune evasion by the parasites could be directly related to parasite factors, (e.g., antigenic variation), or indirectly through the induction of suppressor cells following infection. Several cell types, including suppressive macrophages, myeloid-derived suppressor cells (MDSCs), and regulatory T cells have been shown to contribute to immunosuppression in African trypanosomiasis. In this review, we discuss the key factors that contribute to immunity and immunosuppression during T. congolense infection, and how these factors could aid immune evasion by African trypanosomes. Understanding the regulatory mechanisms that influence resistance and/or susceptibility during African trypanosomiasis could be beneficial in designing effective vaccination and therapeutic strategies against the disease.

NK Cells Are Critical for Optimal Immunity to Experimental Trypanosoma congolense Infection.

NK cells are key innate immune cells that play critical roles in host defense. Although NK cells have been shown to regulate immunity to some infectious diseases, their role in immunity to Trypanosoma congolense has not been investigated. NK cells are vital sources of IFN-γ and TNF-α; two key cytokines that are known to play important roles in resistance to African trypanosomes. In this article, we show that infection with T. congolense leads to increased levels of activated and functional NK cells in multiple tissue compartments. Systemic depletion of NK cells with anti-NK1.1 mAb led to increased parasitemia, which was accompanied by significant reduction in IFN-γ production by immune cells in the spleens and liver of infected mice. Strikingly, infected NFIL3-/- mice (which genetically lack NK cell development and function) on the normally resistant background were highly susceptible to T. congolense infection. These mice developed fulminating and uncontrolled parasitemia and died significantly earlier (13 ± 1 d) than their wild-type control mice (106 ± 26 d). The enhanced susceptibility of NFIL3-/- mice to infection was accompanied by significantly impaired cytokine (IFN-γ and TNF-α) response by CD3+ T cells in the spleens and liver. Adoptive transfer of NK cells into NFIL3-/- mice before infection rescued them from acute death in a perforin-dependent manner. Collectively, these studies show that NK cells are critical for optimal resistance to T. congolense, and its deficiency leads to enhanced susceptibility in infected mice.

Diminazene aceturate (Berenil) downregulates Trypanosoma congolense-induced proinflammatory cytokine production by altering phosphorylation of MAPK and STAT proteins.

Diminazene aceturate (Berenil) is the most commonly used trypanolytic agent in livestock. We previously showed that Berenil downregulates Trypanosoma congolense (T. congolense)-induced cytokine production in macrophages both in vitro and in vivo. Here, we investigated the molecular mechanisms through which the drug alters T. congolense-induced cytokine production in macrophages. We show that pretreatment of macrophages with Berenil significantly downregulated T. congolense-induced phosphorylation of mitogen-activated protein kinase (p38), signal transducer and activator of transcription (STAT) proteins including STAT1 and STAT3, and NFκB activity both in vitro and in vivo. Collectively, our results reveal a mechanistic insight through which Berenil downregulates T. congolense-induced cytokine production in macrophages by inhibiting key signaling molecules and pathways associated with proinflammatory cytokine production.

Myeloid-Derived Suppressor Cells Contribute to Susceptibility to Trypanosoma congolense Infection by Suppressing CD4+ T Cell Proliferation and IFN-γ Production.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of bone marrow-derived myeloid cells that have immune-suppressive activities. These cells have been reported to suppress T cell immunity against tumors as well as in some parasitic and bacterial infections. However, their role during Trypanosoma congolense infection has not been studied. Given that immunosuppression is a hallmark of African trypanosomiasis, we investigated the role of MDSCs in immunity to T. congolense infection. We found increased numbers of MDSCs in the spleen and liver of infected mice, which correlated with increased parasitemia. Depletion of MDSCs significantly increased the percentage of proliferating and IFN-γ-producing CD4+ T cells from the spleen of T. congolense-infected mice. Furthermore, MDSCs from T. congolense-infected mice directly suppressed CD4+ T cell proliferation in a coculture setting. This suppressive effect was abolished by the arginase-1 inhibitor, Nω-hydroxy-nor-l-arginine (nor-NOHA), indicating that MDSCs suppress CD4+ T cell proliferation and function in an arginase-1-dependent manner. Indeed, depletion of MDSCs during infection led to control of the first wave of parasitemia and prolonged survival of infected mice. This was also associated with increased CD4+ T cell proliferation and IFN-γ production. Taken together, our findings identify an important role of MDSCs in the pathogenesis of experimental T. congolense infection via suppression of T cell proliferative and effector cytokine responses in an arginase-1-dependent manner.

Thymic Stromal Lymphopoietin Is Critical for Regulation of Proinflammatory Cytokine Response and Resistance to Experimental Trypanosoma congolense Infection.

African trypanosomiasis (sleeping sickness) poses serious threat to human and animal health in sub-Saharan Africa. Because there is currently no vaccine for preventing this disease and available drugs are not safe, understanding the mechanisms that regulate resistance and/or susceptibility to the disease could reveal novel targets for effective disease therapy and prevention. Thymic stromal lymphopoietin (TSLP) plays a critical role in driving Th2 immune response. Although susceptibility to experimental Trypanosoma congolense infection in mice is associated with excessive proinflammatory responses due in part to impaired Th2 response, the role of TSLP in resistance to African trypanosomiasis has not been well studied. Here, we investigated whether TSLP is critical for maintaining Th2 environment necessary for survival of T. congolense-infected mice. We observed an increased TSLP level in mice after infection with T. congolense, suggesting a role for this cytokine in resistance to the infection. Indeed, TSLPR-/- mice were more susceptible to T. congolense infection and died significantly earlier than their wild-type (WT) controls. Interestingly, serum levels of IFN-γ and TNF-α and the frequency of IFN-γ- and TNF-α-producing CD4+ T cells in the spleens and liver were significantly higher in infected TSLPR-/- mice than in the WT control mice. Susceptibility was also associated with excessive M1 macrophage activation. Treatment of TSLPR-/- mice with anti-IFN-γ mAb during infection abolished their enhanced susceptibility to T. congolense infection. Collectively, our study shows that TSLP plays a critical role in resistance to T. congolense infection by dampening the production of proinflammatory cytokines and its associated M1 macrophage activation.

Hepatic stellate cells regulate liver immunity to visceral leishmaniasis through P110δ-dependent induction and expansion of regulatory T cells in mice.

UNLABELLED: Visceral leishmaniasis (VL) is associated with severe immune dysfunction and if untreated leads to death. Because the liver is one of the primary target organs in VL, unraveling the mechanisms governing the local hepatic immune response is important for understanding the immunopathogenesis of VL. We previously reported that mice with inactivating knockin mutation in the p110δ gene (p110δ(D910A) ) are resistant to VL, due in part to impaired regulatory T-cell (Treg) expansion. In this study, we investigated the mechanism of this resistance by focusing on hepatic stellate cells (HSCs), which are known to regulate Treg induction and expansion. We show that HSCs are infected with Leishmania donovani in vivo and in vitro and that this infection leads to the production of interleukin-2, interleukin-6, and transforming growth factor-ß, cytokines known to induce Tregs. We further demonstrate that L. donovani infection leads to expansion of HSCs in a p110δ-dependent manner and that this correlated with proliferation of hepatic Tregs in vivo. In vitro studies clearly show that L. donovani-infected HSCs induce CD4(+) T cells to become Tregs and expand Tregs in a p110δ-dependent manner. Targeted depletion of HSCs during infection caused a dramatic reduction in liver Treg numbers and proliferation, which was associated with a decrease in interleukin-10 production by hepatic T cells and a more efficient parasite control. CONCLUSION: These results demonstrate the critical role of HSCs in the pathogenesis of VL and suggest that the enhanced resistance of p110δ(D910A) mice to L. donovani infection is due in part to impaired expansion and inability of their HSCs to induce and expand Tregs in the liver.

The B cell adaptor molecule Bam32 is critically important for optimal antibody response and resistance to Trypanosoma congolense infection in mice.

BACKGROUND: Bam32, a 32 kDa adaptor molecule, plays important role in B cell receptor signalling, T cell receptor signalling and antibody affinity maturation in germinal centres. Since antibodies against trypanosome variant surface glycoproteins (VSG) are critically important for control of parasitemia, we hypothesized that Bam32 deficient (Bam32-/-) mice would be susceptible to T. congolense infection. METHODOLOGY/PRINCIPAL FINDINGS: We found that T. congolense-infected Bam32-/- mice successfully control the first wave of parasitemia but then fail to control subsequent waves and ultimately succumb to their infection unlike wild type (WT) C57BL6 mice which are relatively resistant. Although infected Bam32-/- mice had significantly higher hepatomegaly and splenomegaly, their serum AST and ALT levels were not different, suggesting that increased liver pathology may not be responsible for the increased susceptibility of Bam32-/- mice to T. congolense. Using direct ex vivo flow cytometry and ELISA, we show that CD4+ T cells from infected Bam32-/- mice produced significantly increased amounts of disease-exacerbating proinflammatory cytokines (including IFN-γ, TNF-α and IL-6). However, the percentages of regulatory T cells and IL-10-producing CD4+ cells were similar in infected WT and Bam32-/- mice. While serum levels of parasite-specific IgM antibodies were normal, the levels of parasite-specific IgG, (particularly IgG1 and IgG2a) were significantly lower in Bam32-/- mice throughout infection. This was associated with impaired germinal centre response in Bam32-/- mice despite increased numbers of T follicular helper (Tfh) cells. Adoptive transfer studies indicate that intrinsic B cell defect was responsible for the enhanced susceptibility of Bam32-/- mice to T. congolense infection. CONCLUSIONS/SIGNIFICANCE: Collectively, our data show that Bam32 is important for optimal anti-trypanosome IgG antibody response and suppression of disease-promoting proinflammatory cytokines and its deficiency leads to inability to control T. congolense infection in mice.