Interferon-Gamma Promotes Infection of Astrocytes by Trypanosoma cruzi

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ⁺ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.     

Composition of Intestinal Microbiota in Immune-Deficient Mice Kept in Three Different Housing Conditions

Background

Abundance of commensals constituting the intestinal microbiota (IM) affects the immune system and predisposes to a variety of diseases, including intestinal infections, cancer, inflammatory and metabolic disorders. Housing conditions determine the IM and can hence influence the immune system. We analyzed how both variables affect the IM of four immune-compromized mouse lines kept under different housing conditions.

Methodology/Principal Findings

We investigated the IM composition in mice by quantitative 16S rRNA RT-PCR analysis of the main fecal bacterial groups (Enterobacteriaceae, enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella (BP) spp., Clostridium leptum and coccoides groups). Mice were homozygous (HO) or heterozygous (HE) for a targeted inactivating mutation of either the IFN-γ Receptor (R), IFN-γ, Rag1 or IL-4 genes. Overall, differences in IM composition were subtle. However, in the SPF-barrier, total eubacterial loads were higher in Rag1 HE versus Rag1 HO mice as well as in IFN-γR HE versus IFN-γR HO and WT animals. Although absent in WT mice, bifidobacterial loads were higher in HO and HE IFN-γ and Rag1 as well as IL-4 HO mice. Furthermore, BP was slightly lower in HO and HE IFN-γR and IFN-γ mice as well as in IL-4 HO mice as compared to WT controls. Interestingly, IM compositions were comparable in WT mice when kept in individual ventilated cages (IVC) or open cages (OC). IFN-γ HO and HE mice, however, had higher enterobacteria and BP loads, but lacked bifidobacteria when kept in OC versus IVC, as was the case in HO and HE Rag1 mice. In addition, Rag1 HO mice harbored higher clostridial loads when housed in OC as compared to IVC. Unexpectedly, lactobacilli levels were higher in IFN-γR mice when kept in OC versus IVC.

Conclusion/Significance

Housing-dependent and immune-deficiency mediated changes in intestinal microbiota composition were rather subtle but may nevertheless impact immunopathology in experimental models.     

Aggregatibacter actinomycetemcomitans GroEL Protein Promotes Conversion of Human CD4+ T Cells into IFNγ IL10 Producing Tbet+ Th1 Cells

One of the heat shock family protein (Hsp) expressing bacteria is the gram negative, periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). A. actinomycetemcomitans’ Hsp is a 64-kDa GroEL-protein, which has been shown to influence the host cells. In this study we used recombinant A. actinomycetemcomitans GroEL (rAaGroEL) protein as a model antigen to study GroEL-mediated T cell immune response. Human peripheral mononuclear cells (PBMCs), when stimulated with recombinant rAaGroEL, expressed early activation marker CD69 and IL-2R (CD25). CD25 and CD69 expressions were higher in CD4+ T cells compared to CD8+ T cells. rAaGroEL-responding CD4+ T cells expressed IL-10, IFNγ and TNFα cytokines. Interestingly, there were also IL-10 and IFNγ double cytokine producing CD4+ T cells. Additionally, IFNγ expressing CD4+ T cells were also T-bet positive. Altogether the results suggest that rAaGroEL protein affects CD4+ T cells to differentiate into IFNγ IL10-secreting T-bet+ Th1 cells.     

A Statistical Interaction between Circumsporozoite Protein-Specific T Cell and Antibody Responses and Risk of Clinical Malaria Episodes following Vaccination with RTS,S/AS01E

The candidate malaria vaccine RTS,S/AS01_E provides significant but partial protection from clinical malaria. On in vitro circumsporozoite protein (CSP) peptide stimulation and intra-cellular cytokine staining of whole blood taken from 407 5–17 month-old children in a phase IIb trial of RTS,S/AS01_E, we identified significantly increased frequencies of two CSP-specific CD4+ T cells phenotypes among RTS,S/AS01_E vaccinees (IFNγ-IL2+TNF− and IFNγ-IL2+TNF+ CD4+ T cells), and increased frequency of IFNγ-IL2-TNF+ CD4+ T cells after natural exposure. All these T cells phenotypes were individually associated with reductions in the risk of clinical malaria, but IFNγ-IL2-TNF+ CD4+ T cells independently predicted reduced risk of clinical malaria on multi-variable analysis (HR = 0.29, 95% confidence intervals 0.15–0.54, p<0.0005). Furthermore, there was a strongly significant synergistic interaction between CSP-specific IFNγ-IL2-TNF+ CD4+ T cells and anti-CSP antibodies in determining protection against clinical malaria (p = 0.002). Vaccination strategies that combine potent cellular and antibody responses may enhance protection against malaria.     

New Microbicidal Functions of Tracheal Glands: Defective Anti-Infectious Response to Pseudomonas aeruginosa in Cystic Fibrosis

Tracheal glands (TG) may play a specific role in the pathogenesis of cystic fibrosis (CF), a disease due to mutations in the cftr gene and characterized by airway inflammation and Pseudomonas aeruginosa infection. We compared the gene expression of wild-type TG cells and TG cells with the cftr ΔF508 mutation (CF-TG cells) using microarrays covering the whole human genome. In the absence of infection, CF-TG cells constitutively exhibited an inflammatory signature, including genes that encode molecules such as IL-1α, IL-β, IL-32, TNFSF14, LIF, CXCL1 and PLAU. In response to P. aeruginosa, genes associated with IFN-γ response to infection (CXCL10, IL-24, IFNγR2) and other mediators of anti-infectious responses (CSF2, MMP1, MMP3, TLR2, S100 calcium-binding proteins A) were markedly up-regulated in wild-type TG cells. This microbicidal signature was silent in CF-TG cells. The deficiency of genes associated with IFN-γ response was accompanied by the defective membrane expression of IFNγR2 and altered response of CF-TG cells to exogenous IFN-γ. In addition, CF-TG cells were unable to secrete CXCL10, IL-24 and S100A8/S100A9 in response to P. aeruginosa. The differences between wild-type TG and CF-TG cells were due to the cftr mutation since gene expression was similar in wild-type TG cells and CF-TG cells transfected with a plasmid containing a functional cftr gene. Finally, we reported an altered sphingolipid metabolism in CF-TG cells, which may account for their inflammatory signature. This first comprehensive analysis of gene expression in TG cells proposes a protective role of wild-type TG against airborne pathogens and reveals an original program in which anti-infectious response was deficient in TG cells with a cftr mutation. This defective response may explain why host response does not contribute to protection against P. aeruginosa in CF.     

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function

The irreversible Bruton''s tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.     

Leishmania Specific CD4 T Cells Release IFNγ That Limits Parasite Replication in Patients with Visceral Leishmaniasis

Visceral leishmaniasis (VL) is associated with increased circulating levels of multiple pro-inflammatory cytokines and chemokines, including IL-12, IFNγ, and TNFα, and elevated expression of IFNγ mRNA in lesional tissue such as the spleen and bone marrow. However, an immunological feature of VL patients is that their peripheral blood mononuclear cells (PBMCs) typically fail to respond to stimulation with leishmanial antigen. Unexpectedly, it was recently shown that Leishmania specific IFNγ, can readily be detected when a whole blood stimulation assay (WBA) is used. We sought to define the conditions that permit whole blood cells to respond to antigen stimulation, and clarify the biological role of the IFNγ found to be released by cells from VL patients. CD4+ T cells were found to be crucial for and the main source of the IFNγ production in Leishmania stimulated whole blood (WB) cultures. Complement, antibodies and red blood cells present in whole blood do not play a significant role in the IFNγ response. The IFNγ production was reduced by blockade of human leukocyte antigen (HLA)-DR, indicating that the response to leishmanial antigens observed in WB of active VL patients is a classical HLA- T cell receptor (TCR) driven reaction. Most importantly, blockade of IFNγ in ex-vivo splenic aspirate cultures demonstrated that despite the progressive nature of their disease, the endogenous IFNγ produced in patients with active VL serves to limit parasite growth.     

Iron Prevents the Development of Experimental Cerebral Malaria by Attenuating CXCR3-Mediated T Cell Chemotaxis

Cerebral malaria is a severe neurological complication of Plasmodium falciparum infection. Previous studies have suggested that iron overload can suppress the generation of a cytotoxic immune response; however, the effect of iron on experimental cerebral malaria (ECM) is yet unknown. Here we determined that the incidence of ECM was markedly reduced in mice treated with iron dextran. Protection was concomitant with a significant decrease in the sequestration of CD4⁺ and CD8⁺ T cells within the brain. CD4⁺ T cells demonstrated markedly decreased CXCR3 expression and had reduced IFNγ-responsiveness, as indicated by mitigated expression of IFNγR2 and T-bet. Additional analysis of the splenic cell populations indicated that parenteral iron supplementation was also associated with a decrease in NK cells and increase in regulatory T cells. Altogether, these results suggest that iron is able to inhibit ECM pathology by attenuating the capacity of T cells to migrate to the brain.     

NK-, NKT- and CD8-Derived IFNγ Drives Myeloid Cell Activation and Erythrophagocytosis,Resulting in Trypanosomosis-Associated Acute Anemia

African trypanosomes are the causative agents of Human African Trypanosomosis (HAT/Sleeping Sickness) and Animal African Trypanosomosis (AAT/Nagana). A common hallmark of African trypanosome infections is inflammation. In murine trypanosomosis, the onset of inflammation occurs rapidly after infection and is manifested by an influx of myeloid cells in both liver and spleen, accompanied by a burst of serum pro-inflammatory cytokines. Within 48 hours after reaching peak parasitemia, acute anemia develops and the percentage of red blood cells drops by 50%. Using a newly developed in vivo erythrophagocytosis assay, we recently demonstrated that activated cells of the myeloid phagocytic system display enhanced erythrophagocytosis causing acute anemia. Here, we aimed to elucidate the mechanism and immune pathway behind this phenomenon in a murine model for trypanosomosis. Results indicate that IFNγ plays a crucial role in the recruitment and activation of erythrophagocytic myeloid cells, as mice lacking the IFNγ receptor were partially protected against trypanosomosis-associated inflammation and acute anemia. NK and NKT cells were the earliest source of IFNγ during T. b. brucei infection. Later in infection, CD8+ and to a lesser extent CD4+ T cells become the main IFNγ producers. Cell depletion and transfer experiments indicated that during infection the absence of NK, NKT and CD8+ T cells, but not CD4+ T cells, resulted in a reduced anemic phenotype similar to trypanosome infected IFNγR-/- mice. Collectively, this study shows that NK, NKT and CD8+ T cell-derived IFNγ is a critical mediator in trypanosomosis-associated pathology, driving enhanced erythrophagocytosis by myeloid phagocytic cells and the induction of acute inflammation-associated anemia.     

Differential Induction of Antimicrobial REGIII by the Intestinal Microbiota and Bifidobacterium breve NCC2950

The intestinal microbiota is a key determinant of gut homeostasis, which is achieved, in part, through regulation of antimicrobial peptide secretion. The aim of this study was to determine the efficiency by which members of the intestinal microbiota induce the antimicrobial peptide REGIII and to elucidate the underlying pathways. We showed that germfree mice have low levels of REGIII-γ in their ileum and colon compared to mice with different intestinal microbiota backgrounds. Colonization with a microbiota of low diversity (altered Schaedler flora) did not induce the expression of REGIII-γ as effectively as a complex community (specific pathogen free). Monocolonization with the probiotic Bifidobacterium breve, but not with the nonprobiotic commensal Escherichia coli JM83, upregulated REGIII-γ expression. Induction of REGIII-γ by B. breve was abrogated in mice lacking MyD88 and Ticam1 signaling. Both live and heat-inactivated B. breve but not spent culture medium from B. breve induced the expression of REGIII-α, the human ortholog and homolog of REGIII-γ, in human colonic epithelial cells (Caco-2). Taken together, the results suggest that REGIII-γ expression in the intestine correlates with the richness of microbiota composition. Also, specific bacteria such as Bifidobacterium breve NCC2950 effectively induce REGIII production in the intestine via the MyD88-Ticam1 pathway. Treatment with this probiotic may enhance the mucosal barrier and protect the host from infection and inflammation.     

Gene-targeted metagenomic analysis of glucan-branching enzyme gene profiles among human and animal fecal microbiota

Glycoside hydrolases (GHs), the enzymes that breakdown complex carbohydrates, are a highly diversified class of key enzymes associated with the gut microbiota and its metabolic functions. To learn more about the diversity of GHs and their potential role in a variety of gut microbiomes, we used a combination of 16S, metagenomic and targeted amplicon sequencing data to study one of these enzyme families in detail. Specifically, we employed a functional gene-targeted metagenomic approach to the 1-4-α-glucan-branching enzyme (gBE) gene in the gut microbiomes of four host species (human, chicken, cow and pig). The characteristics of operational taxonomic units (OTUs) and operational glucan-branching units (OGBUs) were distinctive in each of hosts. Human and pig were most similar in OTUs profiles while maintaining distinct OGBU profiles. Interestingly, the phylogenetic profiles identified from 16S and gBE gene sequences differed, suggesting the presence of different gBE genes in the same OTU across different vertebrate hosts. Our data suggest that gene-targeted metagenomic analysis is useful for an in-depth understanding of the diversity of a particular gene of interest. Specific carbohydrate metabolic genes appear to be carried by distinct OTUs in different individual hosts and among different vertebrate species'' microbiomes, the characteristics of which differ according to host genetic background and/or diet.     

Tumor Progression Locus 2 Promotes Induction of IFNλ, Interferon Stimulated Genes and Antigen-Specific CD8+ T Cell Responses and Protects against Influenza Virus

Mitogen-activated protein kinase (MAP) cascades are important in antiviral immunity through their regulation of interferon (IFN) production as well as virus replication. Although the serine-threonine MAP kinase tumor progression locus 2 (Tpl2/MAP3K8) has been implicated as a key regulator of Type I (IFNα/β) and Type II (IFNγ) IFNs, remarkably little is known about how Tpl2 might contribute to host defense against viruses. Herein, we investigated the role of Tpl2 in antiviral immune responses against influenza virus. We demonstrate that Tpl2 is an integral component of multiple virus sensing pathways, differentially regulating the induction of IFNα/β and IFNλ in a cell-type specific manner. Although Tpl2 is important in the regulation of both IFNα/β and IFNλ, only IFNλ required Tpl2 for its induction during influenza virus infection both in vitro and in vivo. Further studies revealed an unanticipated function for Tpl2 in transducing Type I IFN signals and promoting expression of interferon-stimulated genes (ISGs). Importantly, Tpl2 signaling in nonhematopoietic cells is necessary to limit early virus replication. In addition to early innate alterations, impaired expansion of virus-specific CD8⁺ T cells accompanied delayed viral clearance in Tpl2^-/- mice at late time points. Consistent with its critical role in facilitating both innate and adaptive antiviral responses, Tpl2 is required for restricting morbidity and mortality associated with influenza virus infection. Collectively, these findings establish an essential role for Tpl2 in antiviral host defense mechanisms.     

A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation

The CD34⁺ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8⁺ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8⁺ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality.     

Reciprocal Regulation of Development of Neutrophil-Dendritic Cell Hybrids in Mice by IL-4 and Interferon-Gamma

Neutrophils contribute to innate host immunity by functioning as professional phagocytes, whereas dendritic cells (DCs) are prototypic antigen presenting cells (APCs) responsible for the induction of adaptive immune responses. We have demonstrated recently that neutrophils trans-differentiate into a unique population, termed “neutrophil-DC hybrids,” expressing surface markers of both neutrophils and DCs and exhibiting dual functionality of both phagocytes and APCs. Although the hybrid cells emerged in significant numbers in murine bone marrow (BM) culture in the presence of GM-CSF, mechanisms regulating their development remained mostly unknown. In this study, we tested a total of 61 cytokines for their potentials to regulate neutrophil-DC hybrid formation using a newly developed BM micro-culture system combined with semi-automated FACS analysis. Several cytokines including GM-CSF were found to promote the generation of neutrophil-DC hybrids defined by the phenotype of CD11c⁺/MHC II⁺/Ly6G⁺. When tested in the presence of GM-CSF, hybrid cell development was enhanced by IL-4 and suppressed by interferon-γ (IFNγ) in dose-dependent fashions. We next determined in vivo impacts of IL-4 and IFNγ on the development of neutrophil-DC hybrids in thioglycollate-induced peritonitis lesions. Intraperitoneal administrations of IL-4/anti-IL-4 antibody complex (IL-4C) significantly increased the number of hybrids recovered from the lesions. By contract, recovery of hybrids was reduced by recombinant IFNγ. With regard to function, those hybrid cells recovered from IL-4C-treated mice and IFNγ-treated mice showed potent abilities to capture E.coli. These observations imply that emergence of neutrophil-DC hybrids in inflammatory sites is tightly regulated by local cytokine milieus.