An Intradermal Inoculation Mouse Model for Immunological Investigations of Acute Scrub Typhus and Persistent Infection

Scrub typhus is a neglected tropical disease, caused by Orientia tsutsugamushi, a Gram-negative bacterium that is transmitted to mammalian hosts during feeding by Leptotrombidium mites and replicates predominantly within endothelial cells. Most studies of scrub typhus in animal models have utilized either intraperitoneal or intravenous inoculation; however, there is limited information on infection by the natural route in murine model skin or its related early host responses. Here, we developed an intradermal (i.d.) inoculation model of scrub typhus and focused on the kinetics of the host responses in the blood and major infected organs. Following ear inoculation with 6 x 10⁴ O. tsutsugamushi, mice developed fever at 11–12 days post-infection (dpi), followed by marked hypothermia and body weight loss at 14–19 dpi. Bacteria in blood and tissues and histopathological changes were detected around 9 dpi and peaked around 14 dpi. Serum cytokine analyses revealed a mixed Th1/Th2 response, with marked elevations of MCP-1/CCL2, MIP-1α/CCL3 and IL-10 at 9 dpi, followed by increased concentrations of pro-inflammatory markers (IL-6, IL-12, IFN-γ, G-CSF, RANTES/CCL5, KC/CCL11, IL-1α/β, IL-2, TNF-α, GM-CSF), as well as modulatory cytokines (IL-9, IL-13). Cytokine levels in lungs had similar elevation patterns, except for a marked reduction of IL-9. The Orientia 47-kDa gene and infectious bacteria were detected in several organs for up to 84 dpi, indicating persistent infection. This is the first comprehensive report of acute scrub typhus and persistent infection in i.d.-inoculated C57BL/6 mice. This is a significant improvement over current murine models for Orientia infection and will permit detailed studies of host immune responses and infection control interventions.     

IL-33-Dependent Endothelial Activation Contributes to Apoptosis and Renal Injury in Orientia tsutsugamushi-Infected Mice

Endothelial cells (EC) are the main target for Orientia tsutsugamushi infection and EC dysfunction is a hallmark of severe scrub typhus in patients. However, the molecular basis of EC dysfunction and its impact on infection outcome are poorly understood. We found that C57BL/6 mice that received a lethal dose of O. tsutsugamushi Karp strain had a significant increase in the expression of IL-33 and its receptor ST2L in the kidneys and liver, but a rapid reduction of IL-33 in the lungs. We also found exacerbated EC stress and activation in the kidneys of infected mice, as evidenced by elevated angiopoietin (Ang) 2/Ang1 ratio, increased endothelin 1 (ET-1) and endothelial nitric oxide synthase (eNOS) expression. Such responses were significantly attenuated in the IL-33^-/- mice. Importantly, IL-33^-/- mice also had markedly attenuated disease due to reduced EC stress and cellular apoptosis. To confirm the biological role of IL-33, we challenged wild-type (WT) mice with a sub-lethal dose of O. tsutsugamushi and gave mice recombinant IL-33 (rIL-33) every 2 days for 10 days. Exogenous IL-33 significantly increased disease severity and lethality, which correlated with increased EC stress and activation, increased CXCL1 and CXCL2 chemokines, but decreased anti-apoptotic gene BCL-2 in the kidneys. To further examine the role of EC stress, we infected human umbilical vein endothelial cells (HUVEC) in vitro. We found an infection dose-dependent increase in the expression of IL-33, ST2L soluble ST2 (sST2), and the Ang2/Ang1 ratio at 24 and 48 hours post-infection. This study indicates a pathogenic role of alarmin IL-33 in a murine model of scrub typhus and highlights infection-triggered EC damage and IL-33-mediated pathological changes during the course of Orientia infection.     

A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model

BackgroundScrub typhus is a vector-borne febrile illness caused by Orientia tsutsugamushi transmitted by the bite of Trombiculid mites. O. tsutsugamushi has a high genetic diversity and is increasingly recognized to have a wider global distribution than previously assumed.Methodology/principle findingsWe evaluated the clinical outcomes and host immune responses of the two most relevant human pathogenic strains of O. tsutsugamushi; Karp (n = 4) and Gilliam (n = 4) in a time-course study over 80 days post infection (dpi) in a standardized scrub typhus non-human primate rhesus macaque model. We observed distinct features in clinical progression and immune response between the two strains; Gilliam-infected macaques developed more pronounced systemic infection characterized by an earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. C-reactive protein (CRP) plasma levels, interferon gamma (IFN-γ, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-γ/IL-10 ratios correlated positively with bacterial load in blood, implying activation of the innate immune response and preferential development of a T helper-type 1 immune response. The O. tsutsugamushi-specific immune memory responses in cells isolated from skin and lymph nodes at 80 dpi were more markedly elevated in the Gilliam-infected macaques than in the Karp-infected group. The comparative cytokine response dynamics of both strains revealed significant up-regulation of IFN-γ, tumor necrosis factor (TNF), IL-15, IL-6, IL-18, regulatory IL-1ra, IL-10, IL-8 and granulocyte-colony-stimulating factor (G-CSF). These data suggest that the clinical outcomes and host immune responses to scrub typhus could be associated with counter balancing effects of pro- and anti-inflammatory cytokine-mediated responses.Currently, no data on characterized time-course comparisons of O. tsutsugamushi strains regarding measures of disease severity and immune response is available. Our study provides evidence for the strain-specificity of host responses in scrub typhus, which supports our understanding of processes at the initial inoculation site (eschar), systemic disease progression, protective and/or pathogenic host immune mechanisms and cellular immune memory function.Conclusions/significanceThis study characterised an improved intradermal rhesus macaque challenge model for scrub typhus, whereby the Gilliam strain infection associated with higher disease severity in the rhesus macaque model than the previous Karp strain infection. Difficulties associated with inoculum quantitation for obligate-intracellular bacteria were overcome by using functional inoculum titrations in outbred mice. The Gilliam-based rhesus macaque model provides improved endpoint measurements and contributes towards the identification of correlates of protection for future vaccine development.     

Orientia tsutsugamushi selectively stimulates the C-type lectin receptor Mincle and type 1-skewed proinflammatory immune responses

Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. The lung is a major target organ of infection, displaying type 1-skewed proinflammatory responses. Lung injury and acute respiratory distress syndrome are common complications of severe scrub typhus; yet, their underlying mechanisms remain unclear. In this study, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethal infection in mice, we performed pulmonary differential expression analysis with NanoString. Of 671 genes examined, we found 312 significantly expressed genes at the terminal phase of disease. Mincle (Clec4e) was among the top 5 greatest up-regulated genes, accompanied with its signaling partners, type 1-skewing chemokines (Cxcr3, Ccr5, and their ligands), as well as Il27. To validate the role of Mincle in scrub typhus, we exposed murine bone marrow-derived macrophages (MΦ) to live or inactivated O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that while heat-killed bacteria stimulated transitory Mincle expression, live bacteria generated a robust response in MΦ, which was validated by indirect immunofluorescence and western blot. Notably, infection had limited impact on other tested CLRs or TLRs. Sustained proinflammatory gene expression in MΦ (Cxcl9, Ccl2, Ccl5, Nos2, Il27) was induced by live, but not inactivated, bacteria; infected Mincle^-/- MΦ significantly reduced proinflammatory responses compared with WT cells. Together, this study provides the first evidence for a selective expression of Mincle in sensing O. tsutsugamushi and suggests a potential role of Mincle- and IL-27-related pathways in host responses to severe infection. Additionally, it provides novel insight into innate immune recognition of this poorly studied bacterium.     

Orientia tsutsugamushi Subverts Dendritic Cell Functions by Escaping from Autophagy and Impairing Their Migration

Background

Dendritic cells (DCs) are the most potent antigen-presenting cells that link innate and adaptive immune responses, playing a pivotal role in triggering antigen-specific immunity. Antigen uptake by DCs induces maturational changes that include increased surface expression of major histocompatibility complex (MHC) and costimulatory molecules. In addition, DCs actively migrate to regional lymph nodes and activate antigen-specific naive T cells after capturing antigens. We characterize the functional changes of DCs infected with Orientia tsutsugamushi, the causative agent of scrub typhus, since there is limited knowledge of the role played by DCs in O. tsutsugamushi infection.

Methodology/Principal Finding

O. tsutsugamushi efficiently infected bone marrow-derived DCs and induced surface expression of MHC II and costimulatory molecules. In addition, O. tsutsugamushi induced autophagy activation, but actively escaped from this innate defense system. Infected DCs also secreted cytokines and chemokines such as IL-6, IL-12, MCP5, MIP-1α, and RANTES. Furthermore, in vitro migration of DCs in the presence of a CCL19 gradient within a 3D collagen matrix was drastically impaired when infected with O. tsutsugamushi. The infected cells migrated much less efficiently into lymphatic vessels of ear dermis ex vivo when compared to LPS-stimulated DCs. In vivo migration of O. tsutsugamushi-infected DCs to regional lymph nodes was significantly impaired and similar to that of immature DCs. Finally, we found that MAP kinases involved in chemotactic signaling were differentially activated in O. tsutsugamushi-infected DCs.

Conclusion/Significance

These results suggest that O. tsutsugamushi can target DCs to exploit these sentinel cells as replication reservoirs and delay or impair the functional maturation of DCs during the bacterial infection in mammals.     

Listeria monocytogenes Alters Mast Cell Phenotype,Mediator and Osteopontin Secretion in a Listeriolysin-Dependent Manner

Whilst mast cells participate in the immune defence against the intracellular bacterium Listeria monocytogenes, there is conflicting evidence regarding the ability of L. monocytogenes to infect mast cells. It is known that the pore-forming toxin listeriolysin (LLO) is important for mast cell activation, degranulation and the release of pro-inflammatory cytokines. Mast cells, however, are a potential source of a wide range of cytokines, chemokines and other mediators including osteopontin, which contributes to the clearing of L. monocytogenes infections in vivo, although its source is unknown. We therefore aimed to resolve the controversy of mast cell infection by L. monocytogenes and investigated the extent of mediator release in response to the bacterium. In this paper we show that the infection of bone marrow-derived mast cells by L. monocytogenes is inefficient and LLO-independent. LLO, however, is required for calcium-independent mast cell degranulation as well as for the transient and selective downregulation of cell surface CD117 (c-kit) on mast cells. We demonstrate that in addition to the key pro-inflammatory cytokines TNF-α and IL-6, mast cells release a wide range of other mediators in response to L. monocytogenes. Osteopontin, IL-2, IL-4, IL-13 and granulocyte macrophage colony-stimulating factor (GM-CSF), and chemokines including CCL2, CCL3, CCL4 and CCL5 are released in a MyD88-dependent manner. The wide range of mediators released by mast cells in response to L. monocytogenes may play an important role in the recruitment and activation of a variety of immune cells in vivo. The cocktail of mediators, however, is unlikely to skew the immune response to a particular effector response. We propose that mast cells provide a hitherto unreported source of osteopontin, and may provide an important role in co-ordinating the immune response during Listeria infection.     

Echinococcus multilocularis: inflammatory and regulatory chemokine responses in patients with progressive, stable and cured alveolar echinococcosis

The progressive growth of Echinococcus multilocularis metacestodes and their tissue infiltration will cause organ malfunction and finally failure. In few patients, E. multilocularis metacestode proliferation will spontaneously regress, but little is known about the determinants which may restrain metacestode survival and growth. In this study, chemokine responses were investigated in E. multilocularis patients at different states of infection, i.e. with progressive, stable and cured alveolar echinococcosis (AE). Characteristic chemokine profiles and changes in their production were observed in AE patients and infection-free controls when their peripheral blood cells were cultured with E. multilocularis antigens. The production of CC and CXC chemokines which associate with inflammation (MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5 and GRO-α/CXCL1) was constitutively larger in AE patients than in controls; and the elevated chemokine releases were equal in patients with progressive, stable or cured AE. Cluster analyses identified three distinct chemokine response profiles; chemokines were enhanced, depressed or produced in similar quantities in AE patients and controls. A disparate cellular responsiveness was observed in AE patients to viable E. multilocularis vesicles; cluster 1 (GRO-α/CXCL1, MCP-3/CCL7, MCP-4/CCL13, TARC/CCL17, LARC/CCL20) and cluster 2 chemokines (PARC/CCL18, MDC/CCL22, MIG/CXCL9) were clearly diminished, while cluster 3 chemokines (MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5) augmented. The increased production of inflammatory chemokines in patients even with cured AE could be induced by residual E. multilocularis metacestode lesions which continuously stimulate production of inflammatory chemokines. E. multilocularis metacestodes also suppressed cellular chemokine production in AE patients, and this may constitute an immune escape mechanism which reduces inflammatory host responses, prevents tissue destruction and organ damage, but may also facilitate parasite persistence.     

Chemokines and cytokines in patients with an occult Onchocerca volvulus infection

Repeated ivermectin treatment will clear microfilaria (Mf) of Onchocerca volvulus from skin and eyes of onchocerciasis patients while adult filaria remains alive and reproductive, and such occult O. volvulus infection may persist for years. To investigate the effect of residual adult filaria on the immune response profile, chemokines and cytokines were quantified 1) in onchocerciasis patients who developed an occult O. volvulus infection (Mf-negative) due to repeated ivermectin treatments, 2) patients who became Mf-negative without ivermectin treatments due to missing re-infection, and 3) endemic and non-endemic O. volvulus Mf-negative controls. With occult O. volvulus infection, serum levels of pro-inflammatory chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, MPIF-1/CCL23 and CXCL8/IL-8 enhanced and approached higher concentrations as determined in infection-free controls, whilst regulatory and Th2-type cytokines and chemokines MCP-4/CCL13, MIP-1δ/CCL15, TARC/CCL17 and IL-13 lessened. Levels of Eotaxin-2/CCL24, MCP-3/CCL7 and BCA-1/CXCL13 remained unchanged. At 3 days post-initial ivermectin treatment, MCP-1/CCL2, MCP-4/CCL13, MPIF-1/CCL23 and Eotaxin-2/CCL24 were strongly enhanced, suggesting that monocytes and eosinophil granulocytes have mediated Mf clearance. In summary, with occult and expiring O. volvulus infections the serum levels of inflammatory chemokines enhanced over time while regulatory and Th2-type-promoting cytokines and chemokines lessened; these changes may reflect a decreasing effector cell activation against Mf of O. volvulus, and in parallel, an enhancing inflammatory immune responsiveness.     

The Sbi Protein Contributes to Staphylococcus aureus Inflammatory Response during Systemic Infection

Staphylococcus aureus is an important human pathogen that causes infections that may present high morbidity and mortality. Among its many virulence factors protein A (SpA) and Staphylococcal binding immunoglobulin protein (Sbi) bind the Fc portion of IgG interfering with opsonophagocytosis. We have previously demonstrated that SpA interacts with the TNF-α receptor (TNFR) 1 through each of the five IgG binding domains and induces the production of pro-inflammatory cytokines and chemokines. The IgG binding domains of Sbi are homologous to those of SpA, which allow us to hypothesize that Sbi might also have a role in the inflammatory response induced by S. aureus. We demonstrate that Sbi is a novel factor that participates in the induction of the inflammatory response during staphylococcal infections via TNFR1 and EGFR mediated signaling as well as downstream MAPKs. The expression of Sbi significantly contributed to IL-6 production and modulated CXCL-1 expression as well as neutrophil recruitment to the site of infection, thus demonstrating for the first time its relevance as a pro-inflammatory staphylococcal antigen in an in vivo model.     

Cytokine and chemokine responses to helminth and protozoan parasites and to fungus and mite allergens in neonates,children, adults,and the elderly

Background

In rural sub-Saharan Africa, endemic populations are often infected concurrently with several intestinal and intravascular helminth and protozoan parasites. A specific, balanced and, to an extent, protective immunity will develop over time in response to repeated parasite encounters, with immune responses initially being poorly adapted and non-protective. The cellular production of pro-inflammatory and regulatory cytokines and chemokines in response to helminth, protozoan antigens and ubiquitous allergens were studied in neonates, children, adults and the elderly.

Results

In children schistosomiasis prevailed (33%) while hookworm and Entamoeba histolytica/E. dispar was found in up to half of adults and the elderly. Mansonella perstans filariasis was only present in adults (24%) and the elderly (25%). Two or more parasite infections were diagnosed in 41% of children, while such polyparasitism was present in 34% and 38% of adults and the elderly. Cytokine and chemokine production was distinctively inducible by parasite antigens; pro-inflammatory Th2-type cytokine IL-19 was activated by Entamoeba and Ascaris antigens, being low in neonates and children while IL-19 production enhanced “stepwise” in adults and elderly. In contrast, highest production of MIP-1delta/CCL15 was present in neonates and children and inducible by Entamoeba-specific antigens only. Adults and the elderly had enhanced regulatory IL-27 cytokine responses, with Th2-type chemokines (MCP-4/CCL13, Eotaxin-2/CCL24) and cytokines (IL-33) being notably inducible by helminth- and Entamoeba-specific antigens and fungus-derived allergens. The lower cellular responsiveness in neonates and children highlighted the development of a parasite-specific cellular response profile in response to repeated episodes of exposure and re-infection.

Conclusions

Following repeated exposure to parasites, and as a consequence of host inability to prevent or eliminate intestinal helminth or protozoa infections, a repertoire of immune responses will evolve with lessened pro-inflammatory and pronounced regulatory cytokines and chemokines; this is required for partial parasite control as well as for preventing inadequate and excessive host tissue and organ damage.

     

IL-4 up-regulates epidermal chemotactic,angiogenic, and pro-inflammatory genes and down-regulates antimicrobial genes in vivo and in vitro: Relevant in the pathogenesis of atopic dermatitis

Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Although the pathogenesis of AD is not fully understood, we and others have shown that IL-4 plays a key role. In this study we aimed to identify keratinocyte genes regulated by IL-4 that may play important roles in the pathophysiology of AD. HaCat cells were treated with IL-4 at various concentrations for 24 h, and PCR gene array on inflammation/autoimmunity was performed three times for analysis of differential gene expression. Of all the 370 genes examined, 32 and 53 genes are up- and down-regulated, respectively. Specifically related to AD, chemokines CCL3L1, CCL8, CCL24, CCL25, CCL26, CXCL6 and CXCL16 are up-regulated by IL-4. Pro-inflammatory factors, such as IL-19, IL-20, IL-1α, IL-12Rβ2, IL-25, IL-31RA, OSMR and nitric oxide synthase 2, are also up-regulated. In addition, IL-4 up-regulates VEGFA, a pro-angiogenic factor. In contrast, antimicrobial peptides (AMPs) or factors involved in APM production, such as IFN-κ, S100s, Toll-like receptors, and several chemokines are down-regulated. Similarly IL-4 also down-regulates TNF-α, lymphotoxin-β, an IgE suppressor, TNFSF18, a T-cells function regulator, and the glucocorticoid receptor. On the in vivo level, real-time RT-PCR on the selected genes confirmed that IL-4 up-regulates chemokines, proinflammatory cytokines while it suppresses AMP production related genes in the skin obtained from IL-4 Tg mice. Detailed examination of these genes will delineate their specific roles in chemotaxis, inflammation, angiogenesis and AMP production, all of which may contribute to the development and progression of AD.     

Interactions of Streptococcus iniae with phagocytic cell line

Streptococcus iniae has become one of the most serious aquatic pathogens in the last decade, causing large losses in wild and farmed fish worldwide. There is clear evidence that this pathogen is capable not only of causing serious disease in fish but also of being transferred to and infecting humans. In this study, we investigate the interaction of S. iniae with two murine macrophage cell lines, J774-A1 and RAW 264.7. Cytotoxicity assay demonstrated significant differences between live and UV-light killed IUSA-1 strains. The burst respiratory activity decreased to baseline after 1 and 4 h of exposure for J774-A1 and RAW 264.7, respectively. Immunofluorescent and ultrastructural study of infected cells confirmed the intracellular localization of bacteria at 1 h and 24 h post-infection. Using qRT-PCR arrays, we investigated the changes in the gene expression of immune relevant genes associated with macrophage activation. In this screening, we identified 11 of 84 genes up-regulated, we observed over-expression of pro-inflammatory response as IL-1α, IL-1β, and TNF-α, without a good anti-inflammatory response. Present findings suggest a capacity of S. iniae to modulate a mammalian macrophages cell lines to their survival and replication intracellular, which makes this cell type as a reservoir for continued infection.     

G-protein-coupled receptor independent, immunomodulatory properties of chemokine CXCL9

Certain chemokines possess anti-angiogenic and antibacterial activity, in addition to their ability to recruit leukocytes. Herein, we demonstrate that CXCL9/MIG induces the expression, by a monocytic cell line and peripheral blood mononuclear cells, of a variety of chemokines including CXCL8/IL-8, CCL3/MIP-1α, CCL4/MIP-1β, CCL2/MCP-1 in a pertussis toxin insensitive manner. Similarly, another cationic chemokine CCL20/MIP-3α, but not the non-cationic chemokines CCL2 or CCL3, stimulated monocytic cells to produce substantial amounts of CXCL8 and CCL3. Microarray experiments demonstrated that CXCL9, but not CCL2, induced the expression of hundreds of genes, many of which have known or proposed immunomodulatory functions. Induction of CXCL8 required the p38 and ERK1/2 mitogen-activated protein kinases but not NFκB, JAK-STAT or JNK signaling pathways. These results collectively demonstrate that CXCL9 has immunomodulatory functions that are not mediated through a G-protein coupled receptor and may possess additional roles in host defenses against infection.     

Strong Type 1, but Impaired Type 2, Immune Responses Contribute to Orientia tsutsugamushi-Induced Pathology in Mice

Scrub typhus is a neglected, but important, tropical disease, which puts one-third of the world''s population at risk. The disease is caused by Orientia tsutsugamushi, an obligately intracellular Gram-negative bacterium. Dysregulation in immune responses is known to contribute to disease pathogenesis; however, the nature and molecular basis of immune alterations are poorly defined. This study made use of a newly developed murine model of severe scrub typhus and focused on innate regulators and vascular growth factors in O. tsutsugamushi-infected liver, lungs and spleen. We found no activation or even reduction in base-line expression for multiple molecules (IL-7, IL-4, IL-13, GATA3, ROR-γt, and CXCL12) at 2, 6 and 10 days post-infection. This selective impairment in type 2-related immune responses correlated with a significant activation of the genes for IL-1β, IL-6, IL-10, TNF-α, IFN-γ, as well as CXCR3- and CXCR1-related chemokines in inflamed tissues. The elevated angiopoietin (Ang)-2 expression and Ang-2/Ang-1 ratios suggested excessive inflammation and the loss of endothelial integrity. These alterations, together with extensive recruitment of myeloperoxidase (MPO)-expressing neutrophils and the influx of CD3⁺ T cells, contributed to acute tissue damage and animal death. This is the first report of selective alterations in a panel of immune regulators during early O. tsutsugamushi infection in intravenously inoculated C57BL/6 mice. Our findings shed new light on the pathogenic mechanisms associated with severe scrub typhus and suggest potential targets for therapeutic investigation.     

A novel genetic locus linked to pro-inflammatory cytokines after virulent H5N1 virus infection in mice

Background

Genetic variation in the human population is a key determinant of influenza disease severity. A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic. To identify variant host genes associated with increased virus replication and severe disease, we performed a quantitative trait locus analysis on pro-inflammatory cytokine production 48 hours after intranasal infection with highly pathogenic H5N1 influenza virus.

Results

Pro-inflammatory cytokines CCL2, TNFα and IFN-α, were measured by ELISA in lung homogenates of DBA/2J (D2), C57BL/6J (B6) and 44 different BXD recombinant inbred mouse strains. Virus titer was also assessed in a subset of these animals. CCL2 (8-fold), TNFα (24-fold) and IFN-α (8-fold) concentrations varied significantly among the different BXD RI strains. Importantly, cytokine concentration correlated very well (r =0.86-0.96, P <0.0001) with virus titer suggesting that early cytokine production is due to increased virus infection and replication. Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26). This locus accounted for nearly 20% of the observed phenotypic variation in the BXD population studied. Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13. To study the role of Slc25a13, we obtained Slc25a13 knockout line, but upon challenge with H5N1 influenza virus observed no effect on CCL2 production, or morbidity and mortality.

Conclusion

A novel genetic locus on chromosome 6 modulates early pro-inflammatory cytokine production and virus replication after highly pathogenic influenza virus infection. Candidate genes, Samd9l and Ica1, may be important for the control of influenza virus infection and pathogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1017) contains supplementary material, which is available to authorized users.