Modulation of host immune responses by protozoal DNA

Veterinary Immunology and Immunopathology 72 (1999) 87±94

Modulation of host immune responses by protozoal DNA Wendy C. Browna,*, Carlos E. Suareza, Lisl K.M. Shodaa, D. Mark Estesb a

Washington State University, Department of Veterinary Microbiology and Pathology, Pullman, WA 99164-7040, USA b University of Missouri, Department of Veterinary Pathobiology, Columbia, MO, USA Accepted 22 September 1999

Abstract The pathology caused by acute Babesia bovis infection is similar to that seen in severe human malaria caused by Plasmodium falciparum infection, which is related to dysregulated production of inflammatory cytokines and nitric oxide (NO). We have observed induction of NO, inducible nitric oxide synthase (iNOS) and inflammatory cytokines in macrophages by B. bovis. Furthermore, proliferation of lymphocytes from individuals never exposed to certain protozoal pathogens can be induced by crude protozoal parasite extracts. We have repeatedly observed stimulation of naõÈve PBMC from cattle to antigenic extracts of Babesia bovis. Based on recent studies demonstrating the mitogenicity of bacterial and other non-vertebrate DNAs for murine B cells and macrophages, the mitogenic properties of B. bovis DNA were examined. B. bovis and E. coli DNAs induced proliferation of PBMC and purified B cells from non-exposed cattle. Stimulatory activity was reduced by DNase treatment and methylation with CpG methylase, indicating the presence of stimulatory non-methylated CpG motifs in the B. bovis genome. B. bovis and E. coli DNAs enhanced IgG secretion by cultured B cells, stimulating IgG1 and more strongly, IgG2. Several hexameric CpG immunostimulatory sequences (ISS) active for murine B cells were identified in an 11 kb fragment of B. bovis DNA. An oligodeoxyribonucleotide containing one of these (AACGTT), located in the rhoptry associated protein-1 (rap-1) open reading frame, stimulated B cell proliferation. These studies identify a potential mechanism by which protozoal parasites may modulate host immune responses, leading to consequences such as hypergammaglobulinemia and splenomegaly. These results also support the use of ISS as vaccine adjuvants to enhance Type 1 immune responses in cattle. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Mitogenic DNA; Babesia bovis; CpG motifs; Immunostimulatory sequences (ISS) *

Corresponding author. Tel.:‡1-509-335-6067; fax: ‡1-509-335-8529 E-mail address: [email protected] (W.C. Brown) 0165-2427/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 9 9 ) 0 0 1 2 0 - 8

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1. Introduction Successful parasites have adopted many strategies to facilitate survival. Babesia bovis, a protozoan parasite of cattle that infects exclusively erythrocytes, can cause acute infection that results in mortality. However, cattle that do recover from acute infection remain persistently infected, thereby ensuring parasite survival by providing a reservoir for subsequent arthropod vectored transmission. Thus, from the perspective of the parasite, elicitation of an immune response that would permit host survival would be advantageous. Leukocyte recognition of CpG motifs in DNA of microbial origin is hypothesized to represent an innate immune defense mechanism which would enable discrimination of pathogen from host DNA and trigger a selective immune response at the site of infection (Pisetsky, 1996). In this paper, we review the stimulation of innate immune responses by B. bovis parasites and babesial DNA, that may contribute to protective host immune responses or enhanced pathology. 2. Macrophage activation by Babesia bovis B. bovis causes pathological changes similar to those seen in Plasmodium falciparum malaria (Wright et al., 1988). Cerebral malaria is causally related to dysregulated production of IFN-g and TNF-a in response to infection (Grau et al., 1989). These cytokines are also believed to enhance nitric oxide (NO)-mediated parasiticidal activity (Taylor-Robinson, 1995; Jacobs et al., 1996), but there is also evidence that NO is involved in the pathology of acute malaria and B. bovis infections (Taylor-Robinson, 1995; Gale et al., 1998). To investigate the potential role of bovine macrophages in either microbicidal activity or pathology in response to B. bovis, we have characterized the production of NO and expression of inducible nitric oxide synthase (iNOS) and inflammatory cytokine genes interleukin 12 (IL-12), TNF-a, IL-1b, and IL-15 in macrophages cultured with B. bovis. We and others have previously demonstrated that chemical donors of NO are inhibitory for the in vitro growth of B. bovis (Johnson et al., 1996; Shoda et al., 1998). A membrane fraction of B. bovis prepared from pelleted organelles stimulated NO production by macrophages costimulated with IFN-g (Stich et al., 1998). The levels of NO2 ÿ produced in response to B. bovis were comparable to those in cultures where parasite viability was inhibited by chemical donors (Shoda et al., 1998). This B. bovis membrane fraction and whole, parasitized red blood cells also induced iNOS transcript expression (Stich et al., 1998). In addition, preliminary studies have indicated that the membrane fraction of B. bovis and infected erythrocytes upregulates IL12 p40, IL-12 p35 and TNF-a transcript expression, which is potentiated by the addition of IFN-g (Shoda et al., 1998). The Babesia products responsible for activating macrophages have not been defined. Glycosylphosphatidylinositol (GPI)-anchored polypeptides of P. falciparum and Trypanosoma cruzi were shown to activate murine macrophages, and to specifically induce inflammatory cytokines including IL-1, IL-12 and TNF-a (Bate et al., 1992; Schofield and Hackett, 1993; Camargo et al., 1997a, b). We are currently investigating the induction of cytokines and NO by B. bovis phospholipids and DNA. Preliminary studies with DNA are described in Section 4.

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3. Mitogenic properties of non-vertebrate DNA The interest in studying protozoal DNA as a potential immunomodulatory agent for bovine macrophages and other leukocytes stems largely from studies with bacterial DNA. Bacterial DNA has multiple effects on different leukocyte subsets, acting both directly on B cells and macrophages and indirectly on natural killer (NK) cells and T cells. The net result of DNA activation is enhanced inflammatory cytokine production and B cell survival and immunoglobulin secretion through an antigen-and apparently T cellindependent manner. DNA derived from Mycobacterium tuberculosis was shown to induce tumor regression in a mouse model (reviewed by Pisetsky, 1996). Tumor resistance invoked by mycobacterial DNA correlated with NK cell and macrophage activation, and in vitro studies demonstrated that the DNA stimulated production of the Type 1 IFNs, IFN-a and IFN-b, as well as IFN-g, and activated NK cells. Because mammalian DNA was ineffective at inducing IFNs or activating NK cells, it was presumed that stimulation was induced by specific base sequences and not the DNA backbone itself, which is highly conserved. Verification of this assumption was established using synthetic oligonucleotides derived from the M. bovis DNA sequence that contained a centered immunostimulatory (ISS) motif consisting of a six base sequence (50 pur pur CG pyr pyr 30 ). Both murine and human leukocytes were stimulated by such synthetic oligonucleotides to produce IFNs and activate NK cells, showing that the phenomenon was not restricted to a single species. An unmethylated CpG motif was shown to be required for activity, and the bases immediately flanking the CpG dinucleotide were also important determinants for stimulation. In the literature, the most immunogenic sequences for in vitro responses are AACGTT, GACGTT, and GTCGTT (Krieg et al., 1995; Ballas et al., 1996). Methylation of the cytosine with a specific methylase that targets the CG dinucleotide completely abrogated the stimulatory properties of the oligonucleotides, showing in these studies a requirement for nonmethylated CpG motifs. Vertebrate DNA largely lacks nonmethylated CpG motifs and is not mitogenic for leukocytes. The mechanism of NK cell activation and subsequent IFN-g production by both NK cells and T cells that was induced by bacterial DNA and non-methylated CpG oligonucleotides was shown to be indirect, and effected through the production of IL-12, TNF-a and IFNa/b produced by macrophages and dendritic cells (Ballas et al., 1996; Halpern et al., 1996; Klinman et al., 1996; Chace et al., 1997; Sparwasser et al., 1998). Additional effects of bacterial DNA on antigen presenting cells (APC) include the induction of IL-6, IL-1b and iNOS (Stacey et al., 1996; Sparwasser et al., 1998). The other major immunomodulatory effect of microbial/non-vertebrate DNA is on B cells, and the same CpG motifs that activate macrophages were also shown activate B cells. Bacterial DNA and CpG-containing ISS in oligonucleotides directly activated murine B cells (independent of T cells) to proliferate and secrete enhanced levels of IgM in vitro (Krieg et al., 1995). This polyclonal B cell activation included both small and large B cells and CD5‡ as well as CD5ÿ B cells (Sun et al., 1997). The molecular mechanisms by which bacterial DNA can stimulate B cells and APC are not completely understood. Murine B cells appear to be activated following uptake of the DNA (Krieg et al., 1995), whereas it was reported that human B cells were activated by

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surface binding (Liang et al., 1996). Furthermore, B cell survival and cell cycle entry are promoted by CpG motifs in DNA. The CpG oligonucleotides prevent apoptosis via sustained activation of NFkB and expression of the anti-apoptosis gene, c-myc (Yi et al., 1996, 1998). For macrophages, DNA is apparently ingested by endocytosis or phagocytosis, and enough remains in an intact form so that it can activate NFkB, leading to downstream activation of inflammatory cytokine genes such as IL-1b and TNF-a (Stacey et al., 1996). 4. Mitogenic properties of B. bovis DNA. Because it was shown that DNA from certain non-vertebrate organisms, including nematodes, mollusks, yeast, and insects, caused polyclonal activation of murine Blymphocytes (Sun et al., (1996, 1997)), experiments were performed to determine the mitogenic properties of B. bovis DNA for bovine leukocytes (Brown et al., 1998). In addition, since human B cells paradoxically failed to proliferate in response to E. coli DNA even though they could be stimulated by synthetic CpG oligonucleotides (Liang et al., 1996), E. coli DNA was included as a control. We had repeatedly observed proliferation of PBMC from Babesia-naõÈve cattle in response to crude B. bovis antigen preparations, but the mitogenic components had not been defined. To determine if a mitogenic component could be DNA, crude membrane antigen was incubated with DNase, and compared with untreated or mock-treated samples for stimulation of PBMC from naõÈve and immune cattle. Stimulation of PBMC from cattle never exposed to Babesia by B. bovis membrane antigen was abrogated by DNase treated antigen, whereas the proliferative response by PBMC from immune animals was only partially inhibited. Purified B. bovis and E. coli DNA induced dose-dependent proliferation of bovine Blymphocytes, whereas calf thymus DNA was not stimulatory. The response to B. bovis and E. coli DNA was not inhibited by the LPS inhibitor, polymyxin B sulfate, and the DNA preparations did not contain sufficient amounts of contaminating LPS to cause B cell proliferation. Furthermore, any potential contamination with Mycoplasma sp., which are notorious contaminants of many Plasmodium falciparum stocks (Turrini et al., 1997), was ruled out by a PCR assay. B. bovis and E. coli DNAs also enhanced IgG secretion by cultured B cells, stimulating predominantly IgG2 and, to a lesser extent, IgG1 production. To determine if the mitogenicity of DNA was due to the presence of non-methylated CpG motifs, B. bovis DNA was examined for sensitivity to HpaII digestion, and methylated DNA was assayed for B cell stimulation. Like DNAs from other organisms that were mitogenic for murine B cells (Sun et al., 1997), B. bovis DNA is largely nonmethylated, which is based on its sensitivity to HpaII. As predicted, the mitogenicity of B. bovis and E. coli DNAs was reduced upon methylation, indicating the presence of stimulatory non-methylated CpG motifs in the B. bovis genome. The frequency of CpG dinucleotides in the B. bovis genome was estimated from analysis of an 11 kb fragment of genomic DNA, which contained a relatively low abundance (0.033) of CGs compared to the expected frequency (0.0625). As anticipated, several hexameric CpG immunostimulatory sequences (ISS) active for murine B cells were identified in the 11 kb fragment of B. bovis DNA. A synthetic 12-mer oligonucleotide containing one of these ISS

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(AACGTT), located in the rhoptry associated protein-1 (rap-1) open reading frame, was synthesized and tested for stimulation of B cell proliferation. As controls, identical oligonucleotides containing the CG in reverse orientation (GC), a methylated CG (C*G) or lacking the CG were tested together with two CpG oligonucleotides previously shown to stimulate murine B cells. The CpG oligonucleotides reproducibly stimulated dosedependent proliferative responses, whereas the control oligonucleotides were significantly less effective at stimulating B cell proliferation. Since B. bovis-infected erythrocytes unpregulate macrophage iNOS (Stich et al., 1998), and preliminary studies indicated a similar stimulation of IL-12 and TNF-a expression (Shoda et al., 1998), the regulation of cytokines and iNOS expression by B. bovis and E. coli DNA are also being examined. Preliminary results indicated that B. bovis and E. coli DNAs stimulated modest increases in IL-1b, TNF-a, and IL-15, whereas E. coli but not B. bovis DNA stimulated dramatic IL-12 and iNOS expression (unpubl. observ.). 5. Synopsis Together, these studies demonstrate activation of both macrophages and B cells by babesial parasites, which appears to be mediated by parasite DNA and other components present in organelle- and membrane-enriched fractions of merozoites. Activation of innate immune responses by Babesia during acute infection could contribute to severe pathology associated with an acute inflammatory response, or alternatively recovery from disease and establishment of persistent infection. The timing and magnitude of the innate immune response against these parasites will likely be critical. The acute response in malaria and B. bovis infection, proposed originally by Ian Clark and reviewed by Clark et al. (1991), is linked to an overproduction of inflammatory cytokines, such as TNF-a, which results in a multi-system pathology known as systemic inflammatory response syndrome (SIRS) that includes fever, hypoglycemia, dyserythropoesis, and vascular damage in the gut, lungs and brain. Polyclonal activation of B cells by DNA released by dying parasites could contribute to this pathology and to the splenomegaly and hypergammaglobulinemia characteristic of many protozoal parasitic infections. The potent mitogenic effects of E. coli DNA for bovine macrophages and B cells also support the use of ISS as vaccine adjuvants to enhance Type 1 immune responses in cattle. It has become evident that one of the reasons for the unparalleled immunogenicity of DNA vaccines is that the plasmid vector alone is mitogenic, and stimulates the production of IL-12, IL-18 and Type I IFN in APC (Roman et al., 1997). Recently, the requirement for the presence of a specific ISS (AACGTT) for successful vaccination with intradermal injection of a DNA vaccine encoding b-galactosidase was demonstrated (Sato et al., 1996; Roman et al., 1997). Several studies have also successfully used ISScontaining plasmid DNA or CpG oligonucleotides as adjuvants to enhance IFN-g and IgG2a (Type 1) responses in mice immunized with protein antigen in an adjuvant, such as alum or incomplete Freund'rs adjuvant (Chu et al., 1997; Leclerc et al., 1997; Roman et al., 1997). Used alone, these adjuvants typically induce a default Type 2 response to the protein antigen. The feasibility of similarly enhancing Type 1 immune responses with CpG oligonucleotides in cattle is suggested by our preliminary results showing

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upregulation of IL-12 by E. coli DNA. In cattle, IL-12 is a potent inducer of IFN-g (Brown et al., 1996; Brown and Estes, 1997) and IFN-g enhances opsonizing IgG2 production in both T-independent and T-dependent systems (Estes et al., 1994, 1998; Brown et al., 1999). In addition, Type I IFNs also promote strong IFN-g responses by antigen-activated T cells (Tuo et al., 1999) and promote enhanced IgG2 production by B cells activated via T-dependent means (Estes et al., 1998). We are currently investigating the capability of defined CpG oligonucleotides to enhance production of Type 1 cytokines, IL-12, IL-18, and Type I IFN by bovine macrophages, and to promote enhanced IFN-g and IgG2 responses during priming with soluble protein antigens. Acknowledgements We thank Kim Kegerreis for excellent technical assistance. The author's research reviewed in this paper was supported by the National Institutes of Health Grant R01AI30136 and by the United States Department of Agriculture National Research Initiative Competitive Research Grants 95-37204-2347, 96-35204-3584, 97-35204-4513, and 9835204-6462.

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