Lst1 deficiency has a minor impact on course and outcome of the host response to influenza A H1N1 infections in mice
© Leist et al. 2016
Received: 12 November 2015
Accepted: 19 January 2016
Published: 27 January 2016
Previously, we performed a quantitative trait locus (QTL) mapping study in BXD recombinant inbred mice to identify host genetic factors that confer resistance to influenza A virus infection. We found Lst1 (leukocyte specific transcript 1) as one of the most promising candidate genes in the Qivr17-2 locus because it is non-functional in DBA/2 J mice. Several studies have proposed that LST1 plays a role in the immune response to inflammatory diseases in humans and has additional immune-regulatory functions. Here, we evaluated the relevance of LST1 for the host response to influenza A infection in B6-Lst1 −/− mutant mice.
To investigate the role of LST1, we infected B6-Lst1 −/− mutant and C57BL/6 N wild-type mice with a low-virulent influenza A virus (PR8M; H1N1). Lst1 deficient mice exhibited significantly increased body weight loss at days 5 and 6 after infection and slightly increased lethality compared to infected wild-type mice. Determination of viral loads, histopathological examination and analysis of immune cell composition in bronchoalveolar lavage of infected lungs did not reveal any obvious differences between KO and wild-type mice.
The absence of Lst1 leads to a slightly more susceptible phenotype. However, deletion of Lst1 in DBA/2 J mice alone does not explain the high susceptibility of this strain to PR8M influenza infections.
KeywordsInfluenza A virus Lst1 KO mouse mutant Animal model
Each year, about 500 million people are infected by influenza A virus worldwide, of which about 500,000 die. We and others showed that the genetic background strongly influences the course and outcome of influenza A virus infections in different mouse inbred strains [1–4]. To identify genes that influence resistance or susceptibility to influenza A infections, we performed a genome-wide quantitative mapping study using the BXD recombinant inbred strains derived from C57BL/6 J and DBA/2 J. When infected with a low virulent isolate of PR8 influenza A virus (designated PR8M), DBA/2 J mice are highly susceptible and die within 5–7 days post infection (p.i.), whereas C57BL/6 J mice survive [1, 5]. We found about 30 candidate genes across five QTL regions . One of the most promising candidates in the Qivr17-2 (quantitative trait for influenza virus resistance on chromosome 17) locus was the leukocyte specific transcript 1 (Lst1). The human gene LST1 and its mouse homologue Lst1, formerly described as B144 , are located in the MHC class III locus encoding numerous genes involved in the immune response [7, 8]. Transcripts are most abundant in immune cells, especially B cells, T cells, monocytes and dendritic cells . Lst1 expression has been shown to be up-regulated by inflammatory stimuli [8, 10]. DBA/2 J mice exhibit a deletion in the Lst1 which results in a translational frame shift that most likely causes a premature stop codon and thus results in a truncated, non-functional protein . Expression of Lst1 transcripts was up-regulated in C57BL/6 J mice after infection with influenza A virus (PR8M; H1N1). Expression levels increased already at day 2 post infection (p.i.), showing a peak of expression at day 8 p.i.. At later time points Lst1 expression decreased and reached levels that were similar to non-infected mice on day 18 p.i. [2, 11]. Thus, Lst1 expression was found both during the innate and adaptive phase of the host response to influenza and peaked at the time point of T cell infiltration. Bio-GPS (http://biogps.org) expression studies show that Lst1 was mainly expressed in immune cells including mast cells, macrophages and dendritic cells.
To further characterize the role of LST1 after influenza A infection, we studied the host response in a Lst1 mouse knock-out (KO) model. The KO strain C57BL/6 N-Lst1 tm1(KOMP)Vlcg was created from the ES cell clone 12118A-B1 (obtained from the KOMP Repository; www.komp.org). It harbors a reporter-tagged deletion of the Lst1 gene. We confirmed insertion of the targeting cassette into the coding region of exon two and three by PCR genotyping (Additional file 1: Figure S1A) and showed absence of transcripts in knock-out mice by reverse transcription PCR (Additional file 1: Figure S1B).
In conclusion, B6-Lst1 −/− KO mice are slightly more susceptible to PR8M influenza A infection which is reflected in an increased body weight loss and slightly reduced survival rate. The susceptibility of KO mice is much less pronounced compared to DBA/2 J mice. Thus, our results show some contribution of Lst1 to susceptibility of DBA/2 J mice. However, its effect is rather small. Therefore, other genes with polymorphisms in Qivr17-2 as well as polymorphic genes in other QTL regions are contributing to the high susceptibility of DBA/2 J mice. Furthermore, it is conceivable that Lst1 has a major contribution by interacting with additional DBA/2 J alleles in Qivr17-2 or other QTLs that were mapped previously. These results emphasize the influence of different gene loci on the host response to influenza A H1N1 infection in mice.
There are numerous reports describing that LST1 plays an important role in the immune response to inflammatory diseases in humans [10, 13–15], in bacterial infections  and in signal transduction [16, 17]. It has been demonstrated that LST1 plays a crucial role for transmembrane cell to cell communication , which was shown to be important for the intercellular transport of bacteria and retroviruses . Furthermore, the expression of the Lst1 gene was shown to be up-regulated in response to lipopolysaccharide, interferon-γ and bacterial infections .
The LST1 gene has been studied extensively at the gene and mRNA level , but the biological functions of the protein product are largely unknown. Overexpression of LST1 in several human cell lines leads to the formation of filopodia-like membrane protrusions . Recently, it was proposed that LST1 promotes the formation of tunneling nanotubes . Interestingly, it has been shown that several persistent viruses, like HIV and herpes viruses, are able to use those nanotubes for intercellular transfer [22–24]. This mechanism of viral spread has not been demonstrated for influenza A viruses. In addition, it was shown recently that tunneling nanotubes promote networking of immune cells and can mediate transfer of MHC class I molecules between distant cells . This function might be disturbed in Lst1 KO mice leading to a slightly enhanced susceptibility to PR8M influenza A. We found in other mouse knock-out lines that the low-virulent PR8 virus (PR8M, ) is well suitable to detect even small differences in susceptibility. However, it is still possible that Lst1 KO mice may exhibit a larger difference in phenotype when infected with another influenza virus subtype or variant.
To our knowledge the Lst1 knock-out mouse model described here is the first in vivo model investigating the role of LST1 during influenza A infections. However, several open questions still remain with respect to the many biological functions of LST1 in other contexts. The mouse model which we generated will help to elucidate also these other functions.
We would like to thank Peter R.W.A. van Run (Department of Viroscience, Erasmus Medical Center, Rotterdam) for supporting the preparation of the histological slides. This work was supported by intra-mural grants from the Helmholtz-Association (Program Infection and Immunity), a research grant FluResearchNet (No. 01KI07137) and a research grant ‘Infection challenge in the German Mouse Clinic’ from the German Ministry of Education and Research to KS. The work described here was part of a PhD thesis work by SRL at the University of Veterinary Medicine Hannover, Germany.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Srivastava B, Blazejewska P, Hessmann M, Bruder D, Geffers R, Mauel S, et al. Host genetic background strongly influences the response to influenza a virus infections. PLoS One. 2009;4:e4857.PubMed CentralView ArticlePubMedGoogle Scholar
- Nedelko T, Kollmus H, Klawonn F, Spijker S, Lu L, Hessman M, et al. Distinct gene loci control the host response to influenza H1N1 virus infection in a time-dependent manner. BMC Genomics. 2012;13:411.PubMed CentralView ArticlePubMedGoogle Scholar
- Pica N, Iyer A, Ramos I, Bouvier NM, Fernandez-Sesma A, Garcia-Sastre A, et al. The DBA.2 mouse is susceptible to disease following infection with a broad, but limited, range of influenza A and B viruses. J Virol. 2011;85(23):12825–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Boon AC, De Beauchamp J, Hollmann A, Luke J, Kotb M, Rowe S, et al. Host genetic variation affects resistance to infection with a highly pathogenic H5N1 influenza A virus in mice. J Virol. 2009;83:10417–26.PubMed CentralView ArticlePubMedGoogle Scholar
- Blazejewska P, Koscinski L, Viegas N, Anhlan D, Ludwig S, Schughart K. Pathogenicity of different PR8 influenza A virus variants in mice is determined by both viral and host factors. Virology. 2011;412:36–45.View ArticlePubMedGoogle Scholar
- Tsuge I, Shen FW, Steinmetz M, Boyse EA. A gene in the H-2S:H-2D interval of the major histocompatibility complex which is transcribed in B cells and macrophages. Immunogenetics. 1987;26:378–80.View ArticlePubMedGoogle Scholar
- de Baey A, Holzinger I, Scholz S, Keller E, Weiss EH, Albert E. Pvu II polymorphism in the primate homologue of the mouse B144 (LST-1). A novel marker gene within the tumor necrosis factor region. Hum Immunol. 1995;42:9–14.View ArticlePubMedGoogle Scholar
- Holzinger I, de Baey A, Messer G, Kick G, Zwierzina H, Weiss EH. Cloning and genomic characterization of LST1: a new gene in the human TNF region. Immunogenetics. 1995;42:315–22.View ArticlePubMedGoogle Scholar
- de Baey A, Fellerhoff B, Maier S, Martinozzi S, Weidle U, Weiss EH. Complex expression pattern of the TNF region gene LST1 through differential regulation, initiation, and alternative splicing. Genomics. 1997;45:591–600.View ArticlePubMedGoogle Scholar
- Mulcahy H, O’Rourke KP, Adams C, Molloy MG, O’Gara F. LST1 and NCR3 expression in autoimmune inflammation and in response to IFN-gamma, LPS and microbial infection. Immunogenetics. 2006;57:893–903.View ArticlePubMedGoogle Scholar
- Pommerenke C, Wilk E, Srivastava B, Schulze A, Novoselova N, Geffers R, et al. Global transcriptome analysis in influenza-infected mouse lungs reveals the kinetics of innate and adaptive host immune responses. PLoS One. 2012;7:e41169.PubMed CentralView ArticlePubMedGoogle Scholar
- Haller O, Arnheiter H, Lindenmann J. Natural, genetically determined resistance toward influenza virus in hemopoietic mouse chimeras. Role of mononuclear phagocytes. J Exp Med. 1979;150:117–26.View ArticlePubMedGoogle Scholar
- Heidemann J, Kebschull M, Tepasse PR, Bettenworth D. Regulated expression of leukocyte-specific transcript (LST) 1 in human intestinal inflammation. Inflamm Res. 2014;63:513–7.View ArticlePubMedGoogle Scholar
- Mewar D, Marinou I, Lee ME, Timms JM, Kilding R, Teare MD, et al. Haplotype-specific gene expression profiles in a telomeric major histocompatibility complex gene cluster and susceptibility to autoimmune diseases. Genes Immun. 2006;7:625–31.View ArticlePubMedGoogle Scholar
- Nagy GR, Gyorffy B, Galamb O, Molnar B, Nagy B, Papp Z. Use of routinely collected amniotic fluid for whole-genome expression analysis of polygenic disorders. Clin Chem. 2006;52:2013–20.View ArticlePubMedGoogle Scholar
- Lehner B, Semple JI, Brown SE, Counsell D, Campbell RD, Sanderson CM. Analysis of a high-throughput yeast two-hybrid system and its use to predict the function of intracellular proteins encoded within the human MHC class III region. Genomics. 2004;83:153–67.View ArticlePubMedGoogle Scholar
- Stepanek O, Draber P, Horejsi V. Palmitoylated transmembrane adaptor proteins in leukocyte signaling. Cell Signal. 2014;26:895–902.View ArticlePubMedGoogle Scholar
- Schiller C, Diakopoulos KN, Rohwedder I, Kremmer E, von Toerne C, Ueffing M, et al. LST1 promotes the assembly of a molecular machinery responsible for tunneling nanotube formation. J Cell Sci. 2013;126:767–77.View ArticlePubMedGoogle Scholar
- Davis DM, Sowinski S. Membrane nanotubes: dynamic long-distance connections between animal cells. Nat Rev Mol Cell Biol. 2008;9:431–6.View ArticlePubMedGoogle Scholar
- Schiller C, Nowak C, Diakopoulos KN, Weidle UH, Weiss EH. An upstream open reading frame regulates LST1 expression during monocyte differentiation. PLoS One. 2014;9:e96245.PubMed CentralView ArticlePubMedGoogle Scholar
- Raghunathan A, Sivakamasundari R, Wolenski J, Poddar R, Weissman SM. Functional analysis of B144/LST1: a gene in the tumor necrosis factor cluster that induces formation of long filopodia in eukaryotic cells. Exp Cell Res. 2001;268:230–44.View ArticlePubMedGoogle Scholar
- Eugenin EA, Gaskill PJ, Berman JW. Tunneling nanotubes (TNT) are induced by HIV-infection of macrophages: a potential mechanism for intercellular HIV trafficking. Cell Immunol. 2009;254:142–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Sherer NM, Lehmann MJ, Jimenez-Soto LF, Horensavitz C, Pypaert M, Mothes W. Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission. Nat Cell Biol. 2007;9:310–5.PubMed CentralView ArticlePubMedGoogle Scholar
- Sowinski S, Jolly C, Berninghausen O, Purbhoo MA, Chauveau A, Kohler K, et al. Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nat Cell Biol. 2008;10:211–9.View ArticlePubMedGoogle Scholar
- Schiller C, Huber JE, Diakopoulos KN, Weiss EH. Tunneling nanotubes enable intercellular transfer of MHC class I molecules. Hum Immunol. 2013;74:412–6.View ArticlePubMedGoogle Scholar
- Rimmelzwaan GF, Kuiken T, van Amerongen G, Bestebroer TM, Fouchier RA, Osterhaus AD. Pathogenesis of influenza A (H5N1) virus infection in a primate model. J Virol. 2001;75:6687–91.PubMed CentralView ArticlePubMedGoogle Scholar