Cloning and characterization of deer mouse (Peromyscus maniculatus) cytokine and chemokine cDNAs
© Schountz et al; licensee BioMed Central Ltd. 2004
Received: 02 October 2003
Accepted: 13 January 2004
Published: 13 January 2004
Sin Nombre virus (SNV) establishes a persistent infection in the deer mouse, Peromyscus maniculatus. A strong antibody response occurs in response to SNV infection, but the role of the innate immune response is unclear. To address this issue, we have initiated an effort to identify and characterize deer mouse cytokine and chemokine genes. Such cytokines and chemokines are involved in various aspects of immunity, including the transition from innate to adaptive responses, type I and type II responses, recruitment of leukocytes to sites of infection, and production of mature cells from bone marrow progenitors.
We established a colony of SNV antibody-negative deer mice and cloned 11 cytokine and chemokine partial cDNA sequences using directed PCR. Most of the deer mouse sequences were highly conserved with orthologous sequences from other rodent species and functional domains were identified in each putative polypeptide.
The availability of these sequences will allow the examination of the role of these cytokines in deer mouse responses to infection with Sin Nombre virus.
Deer mice (Peromyscus maniculatus) are the principal natural host of Sin Nombre virus (SNV), the etiologic agent of most hantavirus pulmonary syndrome (HPS) cases in humans in North America [1–3]. Deer mice are found throughout most of North America, except for the eastern seaboard and southeast United States .
SNV predominantly infects capillary endothelial cells without discernible pathology . A prominent feature of HPS is capillary leakage and subsequent hypotension that causes death by cardiac failure. In addition to resident alveolar macrophages, lymphocytes infiltrate the lungs of patients and contribute to the pathology by secreting proinflammatory cytokines, including interferon-γ (IFNγ), interleukin-2 (IL-2), tumor necrosis factor (TNF), and lymphotoxin (LT) . These characteristics suggest HPS may be a cytokine-mediated immunopathology.
As is usual with most long-term reservoir hosts, deer mice exhibit little pathology during acute infection with SNV . The virus infects capillary endothelial cells of deer mouse lungs, but lymphocyte infiltration or inflammation are not observed. After acute infection, the virus establishes persistence in many tissues  and most, if not all, deer mice remain infected for the remainders of their lives.
The role of the immune response in infected deer mice is unclear because few reagents of defined specificity have been developed to characterize their immune function. A strong IgG antibody response occurs [7, 9], suggesting that B cells and helper T cells participate in containment of the virus, but the role of cytokines has not been characterized. As part of our continuing efforts to develop the deer mouse as a model for SNV research [10, 11]; (Green et al., in press; Richens et al., submitted), we established a colony of deer mice captured in a region where SNV is prevalent [12, 13]. Splenocytes obtained from deer mice from this colony were used to clone several cytokine and chemokine cDNA sequences. These sequences represent several components of the immune response, including the transition from innate to adaptive responses (IL-12a, IL-21, IL-23a), type I (IL-2) and type II responses (IL-6, IL-13), a colony stimulating factor (GM-CSF), and chemokines (CCL2, CCL3, CCL4, CXCL2). The availability of such sequences will permit experimental examination of the roles of these cytokines in deer mice infected with SNV.
Establishment of a deer mouse colony
We identified a location that contained SNV-infected deer mice and trapped 10 deer mice. Nine (4 males and 5 females) had no IgG antibody to SNV at capture, as well as after 36 days of quarantine , and were used to establish the colony. Breeders were kept on a 16:8 light:dark cycle and were provided food and water ad libitum. The colony has been reproductively vigorous and has produced nearly 300 offspring in 3 years.
Pathogenic hantaviruses induce acute inflammatory responses in humans that contribute to HPS disease progression [5, 6]. Much of the pathology occurs because of the production of several inflammatory cytokines in the lungs, including IL-2, IFNγ, tumor necrosis factor (TNF) and lymphotoxin (LT). In addition, pulmonary T cell infiltrates are observed in patients that die from HPS. It is evident that immunopathology contributes to HPS progression, given that SNV does not cause discernible direct cytopathology.
We targeted several deer mouse cytokines important in different aspects of immune responses. Our approach was to identify highly conserved regions of orthologous sequences from rodents and humans and to design primers from them. In many instances no consensus was found, so degenerate primer sets were used. In our analysis we determined the approximate relative sizes of the fragments based upon the known length of M. musculus orthologs; it is likely that some deer mouse genes will be longer or shorter when full-length sequences become available.
We also used cells from ostensibly SNV-susceptible deer mice. It is plausible that, in response to coadaptation with the virus, some selective pressure has occurred over time on deer mouse cytokine genes.
IL-2 is the principal T cell growth factor and is secreted by T helper 1 (Th1) cells during immune responses [30, 31]. It is also produced by pulmonary infiltrates in HPS patients  and probably plays an indirect role in inflammation by augmenting TNF expression . IL-21 is an IL-2 family member that is also secreted by Th1 cells. It was initially described as an activator of NK cells and an augmenter of B cell and T cell proliferation . More recently, it has been shown to play a role in the transition from innate to adaptive responses by limiting NK cell responses and activating cytotoxic T lymphocyte responses . It also skews the immune response towards a type I response by augmenting expression of the T-bet transcription factor, IFNγ and receptors for IL-2, IL-12 and IL-18 .
Although GM-CSF is secreted by Th1 cells , it is commonly used to generate bone marrow-derived dendritic cells (DC). The receptor-binding region of deer mouse GM-CSF is highly conserved with respect to house mouse and rat GM-CSF. This suggested to us that recombinant house mouse GM-CSF (rGM-CSF), which is commercially available, might bind to the deer mouse GM-CSF receptor, and we have obtained preliminary evidence suggesting that it does (data not shown). Human DC have recently been shown to support the replication of Hantaan virus , which is related to SNV. It should be possible to generate deer mouse DC with rGM-CSF to determine whether they can be infected by SNV and to determine whether there is a functional consequence of infection.
Both IL-12a and IL-23a polypeptides form heterodimers with the IL-12b (p40) subunit to form IL-12 and IL-23, respectively [24, 36]. IL-12 facilitates the transition from the innate response to a type I adaptive immune response, while IL-23 appears to sustain proliferation of Th1 cells during the course of an immune response [24, 37]. Both are produced by macrophages and dendritic cells and influence T cell maturation towards a Th1 phenotype.
In addition to Th2 cells, IL-6 is expressed by many other cell types and has broad biological activity, including a major role in augmenting antibody production in activated B cells . IL-13 is closely linked to the IL-4 gene cluster in humans and house mice and has a role in class switching and controlling inflammatory responses [39, 40].
Chemokine production in humans infected with SNV has not been examined in detail. TNF is produced in the lungs of patients that die from HPS  and mononuclear infiltrates are present in pulmonary tissues . Since chemokines are potent recruiters of blood leukocytes into infected tissues, there must be a prominent role for these proteins in HPS. Deer mice exhibit no conspicuous recruitment of leukocytes into the lungs , thus comparison of chemokine responses in humans and deer mice infected with SNV may provide clues as to how their respective immune systems respond to the virus and how the virus evades the immune response in deer mice to establish persistence.
We did not find any conspicuous differences in the amino acid sequences of deer mouse cytokines or chemokines compared to rat or house mouse orthologs that would suggest a functional difference in the mechanism by which these molecules exert their effects in vivo. More likely, immunological decisions are made during acute infection in deer mice that lead to a qualitative difference in cytokine profiles. These sequences and others that we previously reported  will be useful in characterizing cytokine and chemokine responses in deer mice. By comparing the profiles of these responses in deer mice and humans, it is possible that therapeutic targets can be identified in humans infected with hantaviruses. For HPS, treatment strategies must function quickly because of the rapidity of patient decline; death can occur within hours after medical treatment is sought [5, 41]. Although antiviral drugs may be developed against the virus, it is likely that an immunomodulatory approach that prevents or reverses pulmonary inflammation would be useful in treating patients.
We believe that the primer sets developed in this work can be used to amplify orthologous sequences from a variety of rodent species. Deer mice are New World rodents, while house mice and rats (Rattus sp.) are Old World rodents. Deer mice are about 50 million years divergent (myd) from house mice, while house mice and the laboratory rat (R. rattus) are only 15–25 myd . We attempted to perform phylogenetic analyses; however, the short fragments representing most of the genes possessed insufficient data for meaningful interpretation. We are currently pursuing additional sequence data to address this deficiency (Palmer et al., manuscript in preparation).
We have cloned a number of deer mouse cytokine and chemokine cDNA sequences. These represent several components of immune responses, including transition from innate to adaptive immunity, type I and type II responses, chemotaxis, and a cytokine useful for producing mature monocytic cells from bone marrow. The availability of these sequences will allow the characterization of a portion of the cytokine and chemokine responses in deer mice acutely or persistently infected with SNV.
Establishment of deer mouse colony
Deer mice were trapped under license from the Colorado Division of Wildlife. Sherman live traps were set at a site where deer mice are known to occur (N 38° 59' 18.9", W 108° 17' 13.3")  and baited as described elsewhere . Ten captured deer mice (P. maniculatus nebrascensis) were trapped and bled from the retro-orbital capillary beds and tested for IgG antibody by a standard enzyme-linked immunosorbent assay (ELISA) . Each deer mouse was quarantined outdoors in the shade in buried (10 cm) 20 L plastic buckets with ventilated sealed lids, bedding, lab mouse chow, and apple slices for water and inspected daily. None of the animals died during the quarantine period. One deer mouse was seropositive at capture and was discarded. At day 30 of quarantine, the deer mice were again bled and tested for IgG antibody to SNV by ELISA and still were seronegative. On day 36 of the quarantine, the 9 uninfected deer mice were transported to Mesa State College where they were used to establish a colony with approval of the Institutional Animal Care and Use Committee.
Cloning and sequencing of deer mouse cytokines and chemokines by directed RT-PCR
Deer mouse cytokine and chemokine cDNA sequences compared to house mouse, rat, and human orthologs.
Similarity (%) to:2
Est. coding region (%)1
Primer sets used to clone deer mouse cDNA sequences.
ATG TAC AGC AKG CAG CTC GC
TGT TGA GAT GRY RCT TTG AC
GAG RGR AGA CTT CAC AGA GG
CAG GAT ATR TTT TCT GAC CAC AG
RAC CAC CTC AST TYG GCC AG
TGG TAC ATC TTC AAG TCY TC
CAA YRG CAG CAT GGT ATG GAG
STG GGC YAC YTC GAT TTT GG
GTA GTC ATC TTC TTG GGG AC
CTT TCT AGG AAT TCT TTG GG
AGC CAG ATC TGA GAA GCA GG
CTG CTC CRT GGG CAA AGA CC
GTA GAK GCC ATC AAA GAA GC
AGG CRC CMT TGA GTT TGG TG
ATC ACC AGC AGC ARG TGT CC
RRT CAC ACT AGT TCW CTG TC
SAG ACC AGC AGC CTT TGC TC
RRT GTG GCT ACT TGG CAG C
ACC ATG AAG CTC TGC CTG TC
RTA CTC ATT GAC CCA GGG C
GAC RGA AGT CAT AGC CAC TC
TCA GGW ACG ATC CAG GCT TC
Accession numbers of polypeptide sequences used for alignments in this work.
List of Abbreviations
granulocyte macrophage-colony stimulating factor
Sin Nombre virus
tumor necrosis factor
We are grateful to R. Mackie for assistance during the trapping of the deer mice and A. D~N. Palmer for preliminary phylogenetic analyses. This work was supported by funds from the βββ Biological Honor Society (BD, RG, AB, TR), and the Mesa State College School of Natural Sciences and Mathematics (TS). Additional funding was provided by NIH grant AI25489.
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