G-CSF induction early in uropathogenic Escherichia coli infection of the urinary tract modulates host immunity

Uropathogenic Escherichia coli (UPEC), the causative agent of approximately 85% of urinary tract infections (UTI), is a major health concern primarily affecting women. During infection, neutrophils infiltrate the bladder, but the mechanism of recruitment is not well understood. Here, we investigated the role of UPEC-induced cytokine production in neutrophil recruitment and UTI progression. We first examined the kinetics of cytokine expression during UPEC infection of the bladder, and their contribution to neutrophil recruitment. We found that UPEC infection induces expression of several pro-inflammatory cytokines including granulocyte colony-stimulating factor (G-CSF, CSF-3), not previously known to be involved in the host response to UTI. G-CSF induces neutrophil emigration from the bone marrow; these cells are thought to be critical for bacterial clearance during infection. Upon neutralization of G-CSF during UPEC infection, we found fewer circulating neutrophils, decreased neutrophil infiltration into the bladder and, paradoxically, a decreased bacterial burden in the bladder. However, depletion of G-CSF resulted in a corresponding increase in macrophage-activating cytokines, such as monocyte chemotactic protein-1 (MCP-1, CCL-2) and Il-1beta, which may be key in host response to UPEC infection, potentially resolving the paradoxical decreased bacterial burden. Thus, G-CSF acts in a previously unrecognized role to modulate the host inflammatory response during UPEC infection.     

Role of lymphokines in immune response in respiratory viral infections

The present review deals with the analysis of lymphokine response in two main respiratory viral infections: influenza and respiratory syncytial (RS) infection. Immune response in these two diseases has great phenomenological differences. In particular, in RS infection the intensive response of macrophages and epithelial cells, accompanied mainly by the synthesis and secretion of tumor necrosis factor and interleukin 8, is observed. Due to this fact no protective immunity is formed in RS infection, in contrast to influenza. The specific features of immune and lymphokine response in RS infection make it difficult to develop protective vaccines. At the same time it is with RS infection that the development of chronic bronchopulmonary diseases is linked. The analysis of the role of lymphokine response in respiratory viral infections, particularly in influenza and RS infections, confirms the necessity of revising therapy both in the acute and subacute stages of these diseases.     

H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice

Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition, and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. Primary mouse and human macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro. These results together indicate that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection.     

Influenza A virus infection results in a robust, antigen-responsive, and widely disseminated Foxp3+ regulatory T cell response

Foxp3(+) CD4(+) regulatory T cells (Tregs) represent a highly suppressive T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, although the role of these cells in acute viral infections is poorly understood. The present study sought to examine the induction of Foxp3(+) CD4(+) Tregs in a nonlethal murine model of pulmonary viral infection by the use of the prototypical respiratory virus influenza A. We establish that influenza A virus infection results in a robust Foxp3(+) CD4(+) T cell response and that regulatory T cell induction at the site of inflammation precedes the effector T cell response. Induced Foxp3(+) CD4(+) T cells are highly suppressive ex vivo, demonstrating that influenza virus-induced Foxp3(+) CD4(+) T cells are phenotypically regulatory. Influenza A virus-induced regulatory T cells proliferate vigorously in response to influenza virus antigen, are disseminated throughout the site of infection and primary and secondary lymphoid organs, and retain Foxp3 expression in vitro, suggesting that acute viral infection is capable of inducing a foreign-antigen-specific Treg response. The ability of influenza virus-induced regulatory T cells to suppress antigen-specific CD4(+) and CD8(+) T cell proliferation and cytokine production correlates closely to their ability to respond to influenza virus antigens, suggesting that virus-induced Tregs are capable of attenuating effector responses in an antigen-dependent manner. Collectively, these data demonstrate that primary acute viral infection is capable of inducing a robust, antigen-responsive, and suppressive regulatory T cell response.     

Group 2 Innate Lymphoid Cell Production of IL-5 Is Regulated by NKT Cells during Influenza Virus Infection

Respiratory virus infections, such as influenza, typically induce a robust type I (pro-inflammatory cytokine) immune response, however, the production of type 2 cytokines has been observed. Type 2 cytokine production during respiratory virus infection is linked to asthma exacerbation; however, type 2 cytokines may also be tissue protective. Interleukin (IL)-5 is a prototypical type 2 cytokine that is essential for eosinophil maturation and egress out of the bone marrow. However, little is known about the cellular source and underlying cellular and molecular basis for the regulation of IL-5 production during respiratory virus infection. Using a mouse model of influenza virus infection, we found a robust transient release of IL-5 into infected airways along with a significant and progressive accumulation of eosinophils into the lungs, particularly during the recovery phase of infection, i.e. following virus clearance. The cellular source of the IL-5 was group 2 innate lymphoid cells (ILC2) infiltrating the infected lungs. Interestingly, the progressive accumulation of eosinophils following virus clearance is reflected in the rapid expansion of c-kit⁺ IL-5 producing ILC2. We further demonstrate that the enhanced capacity for IL-5 production by ILC2 during recovery is concomitant with the enhanced expression of the IL-33 receptor subunit, ST2, by ILC2. Lastly, we show that NKT cells, as well as alveolar macrophages (AM), are endogenous sources of IL-33 that enhance IL-5 production from ILC2. Collectively, these results reveal that c-kit⁺ ILC2 interaction with IL-33 producing NKT and AM leads to abundant production of IL-5 by ILC2 and accounts for the accumulation of eosinophils observed during the recovery phase of influenza infection.     

Chemotactic and inflammatory responses in the liver and brain are associated with pathogenesis of Rift Valley fever virus infection in the mouse

Rift Valley fever virus (RVFV) is a major human and animal pathogen associated with severe disease including hemorrhagic fever or encephalitis. RVFV is endemic to parts of Africa and the Arabian Peninsula, but there is significant concern regarding its introduction into non-endemic regions and the potentially devastating effect to livestock populations with concurrent infections of humans. To date, there is little detailed data directly comparing the host response to infection with wild-type or vaccine strains of RVFV and correlation with viral pathogenesis. Here we characterized clinical and systemic immune responses to infection with wild-type strain ZH501 or IND vaccine strain MP-12 in the C57BL/6 mouse. Animals infected with live-attenuated MP-12 survived productive viral infection with little evidence of clinical disease and minimal cytokine response in evaluated tissues. In contrast, ZH501 infection was lethal, caused depletion of lymphocytes and platelets and elicited a strong, systemic cytokine response which correlated with high virus titers and significant tissue pathology. Lymphopenia and platelet depletion were indicators of disease onset with indications of lymphocyte recovery correlating with increases in G-CSF production. RVFV is hepatotropic and in these studies significant clinical and histological data supported these findings; however, significant evidence of a pro-inflammatory response in the liver was not apparent. Rather, viral infection resulted in a chemokine response indicating infiltration of immunoreactive cells, such as neutrophils, which was supported by histological data. In brains of ZH501 infected mice, a significant chemokine and pro-inflammatory cytokine response was evident, but with little pathology indicating meningoencephalitis. These data suggest that RVFV pathogenesis in mice is associated with a loss of liver function due to liver necrosis and hepatitis yet the long-term course of disease for those that might survive the initial hepatitis is neurologic in nature which is supported by observations of human disease and the BALB/c mouse model.     

IL-10 from CD4CD25Foxp3CD127 adaptive regulatory T cells modulates parasite clearance and pathology during malaria infection

The outcome of malaria infection is determined, in part, by the balance of pro-inflammatory and regulatory immune responses. Failure to develop an effective pro-inflammatory response can lead to unrestricted parasite replication, whilst failure to regulate this response leads to the development of severe immunopathology. IL-10 and TGF-beta are known to be important components of the regulatory response, but the cellular source of these cytokines is still unknown. Here we have examined the role of natural and adaptive regulatory T cells in the control of malaria infection and find that classical CD4+CD25(hi) (and Foxp3+) regulatory T cells do not significantly influence the outcome of infections with the lethal (17XL) strain of Plasmodium yoelii (PyL). In contrast, we find that adaptive IL-10-producing, CD4+ T cells (which are CD25-, Foxp3-, and CD127- and do not produce Th1, Th2, or Th17 associated cytokines) that are generated during both PyL and non-lethal P. yoelii 17X (PyNL) infections are able to down-regulate pro-inflammatory responses and impede parasite clearance. In summary, we have identified a population of induced Foxp3- regulatory (Tr1) T cells, characterised by production of IL-10 and down regulation of IL-7Ralpha, that modulates the inflammatory response to malaria.     

Pattern recognition molecule mindin promotes intranasal clearance of influenza viruses

The innate immune response is essential for host defense against microbial pathogen infections and is mediated by pattern recognition molecules recognizing pathogen-associated molecular patterns. Our previous work has demonstrated that the extracellular matrix protein mindin functions as a pattern recognition molecule for bacterial pathogens. In this study, we examined the role of mindin in influenza virus infection. We found that intranasal infection of mindin-deficient mice by influenza virus resulted in dramatically increased virus titers in the lung and intranasal cavity of mutant mice. In contrast, lungs from intratracheally infected mindin-deficient mice contained similar influenza virus titers. We showed that mindin interacted with influenza virus particles directly and that mindin-deficient macrophages exhibited impaired activation after influenza virus infection in vitro. Furthermore, intranasal administration of recombinant mindin significantly enhanced the clearance of influenza virus in wild-type mice. Together, these results demonstrate that mindin plays an essential role in the host innate immune response to influenza virus infection and suggest that mindin may be used as an immune-enhancing agent in influenza infection.     

Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

Background

Cigarette smoke has both pro-inflammatory and immunosuppressive effects. Both active and passive cigarette smoke exposure are linked to an increased incidence and severity of respiratory virus infections, but underlying mechanisms are not well defined. We hypothesized, based on prior gene expression profiling studies, that upregulation of pro-inflammatory mediators by short term smoke exposure would be protective against a subsequent influenza infection.

Methods

BALB/c mice were subjected to whole body smoke exposure with 9 cigarettes/day for 4 days. Mice were then infected with influenza A (H3N1, Mem71 strain), and analyzed 3 and 10 days later (d3, d10). These time points are the peak and resolution (respectively) of influenza infection.

Results

Inflammatory cell influx into the bronchoalveolar lavage (BALF), inflammatory mediators, proteases, histopathology, viral titres and T lymphocyte profiles were analyzed. Compared to smoke or influenza alone, mice exposed to smoke and then influenza had more macrophages, neutrophils and total lymphocytes in BALF at d3, more macrophages in BALF at d10, lower net gelatinase activity and increased activity of tissue inhibitor of metalloprotease-1 in BALF at d3, altered profiles of key cytokines and CD4+ and CD8+ T lymphocytes, worse lung pathology and more virus-specific, activated CD8+ T lymphocytes in BALF. Mice smoke exposed before influenza infection had close to 10-fold higher lung virus titres at d3 than influenza alone mice, although all mice had cleared virus by d10, regardless of smoke exposure. Smoke exposure caused temporary weight loss and when smoking ceased after viral infection, smoke and influenza mice regained significantly less weight than smoke alone mice.

Conclusion

Smoke induced inflammation does not protect against influenza infection. In most respects, smoke exposure worsened the host response to influenza. This animal model may be useful in studying how smoke worsens respiratory viral infections.     

Cigarette smoke worsens lung inflammation and impairs resolution of influenza infection in mice

Background

Cigarette smoke has both pro-inflammatory and immunosuppressive effects. Both active and passive cigarette smoke exposure are linked to an increased incidence and severity of respiratory virus infections, but underlying mechanisms are not well defined. We hypothesized, based on prior gene expression profiling studies, that upregulation of pro-inflammatory mediators by short term smoke exposure would be protective against a subsequent influenza infection.

Methods

BALB/c mice were subjected to whole body smoke exposure with 9 cigarettes/day for 4 days. Mice were then infected with influenza A (H3N1, Mem71 strain), and analyzed 3 and 10 days later (d3, d10). These time points are the peak and resolution (respectively) of influenza infection.

Results

Inflammatory cell influx into the bronchoalveolar lavage (BALF), inflammatory mediators, proteases, histopathology, viral titres and T lymphocyte profiles were analyzed. Compared to smoke or influenza alone, mice exposed to smoke and then influenza had more macrophages, neutrophils and total lymphocytes in BALF at d3, more macrophages in BALF at d10, lower net gelatinase activity and increased activity of tissue inhibitor of metalloprotease-1 in BALF at d3, altered profiles of key cytokines and CD4+ and CD8+ T lymphocytes, worse lung pathology and more virus-specific, activated CD8+ T lymphocytes in BALF. Mice smoke exposed before influenza infection had close to 10-fold higher lung virus titres at d3 than influenza alone mice, although all mice had cleared virus by d10, regardless of smoke exposure. Smoke exposure caused temporary weight loss and when smoking ceased after viral infection, smoke and influenza mice regained significantly less weight than smoke alone mice.

Conclusion

Smoke induced inflammation does not protect against influenza infection.In most respects, smoke exposure worsened the host response to influenza. This animal model may be useful in studying how smoke worsens respiratory viral infections.     

T cell-derived IL-10 and its impact on the regulation of host responses during malaria

Despite intense research, malaria still is the one of the most devastating diseases killing more people than any other parasitic infection. In an attempt to control the infection, the host immune system produces a potent pro-inflammatory response. However, this response is also associated with complications, such as severe anaemia, hypoglycaemia and cerebral malaria. This pronounced production of pro-inflammatory cytokines response is a common feature of malaria caused by parasites infecting humans as well as rodents and primates. A balance between pro- and anti-inflammatory responses may be fundamental to the elimination of the parasite without inducing excessive host pathology. IL-10 is a key cytokine that has been shown to have an important regulatory function in establishing this balance in malaria. Here we discuss which cells can produce IL-10 during infection, and present an overview of the evidence showing that T-cell derived IL-10 plays an important role in regulating malaria pathology. Many different subsets of T cells can produce IL-10, however, evidence is accumulating that it is effector Th1 CD4(+) T cells which provide the crucial source that down-regulates inflammatory pathology during blood-stage malaria infections.     

Effect of Staphylococcus enterotoxin B on the concurrent CD8(+) T cell response to influenza virus infection

Bacterial superantigens have potent in vivo effects. Respiratory viral infections are often associated with secondary bacterial infections, raising the likelihood of exposure to bacterial superantigens after the initiation of the anti-viral immune response. In this study, the general and V beta-specific effects of exposure to Staphylococcal enterotoxin B (SEB) during influenza virus infection on both the ongoing acute and the subsequent recall CD8(+) T cell responses were analyzed, using the well-characterized murine influenza model system and tetrameric MHC/peptide reagents to directly identify virus-specific T cells. The results show that although superantigen exposure during the primary viral infection caused delayed viral clearance, there was remarkably little effect of SEB on the magnitude or TCR repertoire of the ongoing cytolytic T cell response or on the recall response elicited by secondary viral infection. Thus, despite the well-characterized immunomodulatory effects of SEB, there was surprisingly little interference with concurrent anti-viral immunity.     

Natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity

Early control of virus replication by the innate immune response is essential to allow time for the generation of a more effective adaptive immune response. As an important component of innate immunity, complement has been shown to be necessary for protection against numerous microbial infections. This study was undertaken to investigate the role of complement in neutralizing influenza virus. Results demonstrated that the classical pathway of complement mediated serum neutralization of influenza virus. Although nonimmune serum neutralized influenza virus, the mechanism of virus neutralization (VN) required antibody, as sera from RAG1-deficient mice lacked VN activity; moreover, purified natural immunoglobulin M (IgM) restored VN activity to antibody-deficient sera. The mechanism of VN by natural IgM and complement was associated with virion aggregation and coating of the viral hemagglutinin receptor; however, viral lysis did not significantly contribute to VN. Additionally, reconstitution of RAG1-deficient mice with natural IgM resulted in delayed morbidity during influenza virus infection. Collectively, these results provide evidence that natural IgM and the early components of the classical pathway of complement work in concert to neutralize influenza virus and that this interaction may have a significant impact on the course of influenza viral pneumonia.     

Pharmacologic Inhibition of COX-1 and COX-2 in Influenza A Viral Infection in Mice

Background

We previously demonstrated that cyclooxygenase (COX)-1 deficiency results in greater morbidity and inflammation, whereas COX-2 deficiency leads to reduced morbidity, inflammation and mortality in influenza infected mice.

Methodology/Principal Findings

We investigated the effects of COX-1 and COX-2 inhibitors in influenza A viral infection. Mice were given a COX-1 inhibitor (SC-560), a COX-2 inhibitor (celecoxib) or no inhibitor beginning 2 weeks prior to influenza A viral infection (200 PFU) and throughout the course of the experiment. Body weight and temperature were measured daily as indicators of morbidity. Animals were sacrificed on days 1 and 4 post-infection and bronchoalveolar lavage (BAL) fluid was collected or daily mortality was recorded up to 2 weeks post-infection. Treatment with SC-560 significantly increased mortality and was associated with profound hypothermia and greater weight loss compared to celecoxib or control groups. On day 4 of infection, BAL fluid cells were modestly elevated in celecoxib treated mice compared to SC-560 or control groups. Viral titres were similar between treatment groups. Levels of TNF-α and G-CSF were significantly attenuated in the SC-560 and celecoxib groups versus control and IL-6 levels were significantly lower in BAL fluid of celecoxib treated mice versus control and versus the SC-560 group. The chemokine KC was significantly lower in SC-560 group versus control.

Conclusions/Significance

Treatment with a COX-1 inhibitor during influenza A viral infection is detrimental to the host whereas inhibition of COX-2 does not significantly modulate disease severity. COX-1 plays a critical role in controlling the thermoregulatory response to influenza A viral infection in mice.     

Characterization of chicken Mda5 activity: regulation of IFN-β in the absence of RIG-I functionality

In mammals, Mda5 and RIG-I are members of the evolutionary conserved RIG-like helicase family that play critical roles in the outcome of RNA virus infections. Resolving influenza infection in mammals has been shown to require RIG-I; however, the apparent absence of a RIG-I homolog in chickens raises intriguing questions regarding how this species deals with influenza virus infection. Although chickens are able to resolve certain strains of influenza, they are highly susceptible to others, such as highly pathogenic avian influenza H5N1. Understanding RIG-like helicases in the chicken is of critical importance, especially for developing new therapeutics that may use these systems. With this in mind, we investigated the RIG-like helicase Mda5 in the chicken. We have identified a chicken Mda5 homolog (ChMda5) and assessed its functional activities that relate to antiviral responses. Like mammalian Mda5, ChMda5 expression is upregulated in response to dsRNA stimulation and following IFN activation of cells. Furthermore, RNA interference-mediated knockdown of ChMda5 showed that ChMda5 plays an important role in the IFN response of chicken cells to dsRNA. Intriguingly, although ChMda5 levels are highly upregulated during influenza infection, knockdown of ChMda5 expression does not appear to impact influenza proliferation. Collectively, although Mda5 is functionally active in the chicken, the absence of an apparent RIG-I-like function may contribute to the chicken's susceptibility to highly pathogenic influenza.     

Innate Immunity and the Inter-exposure Interval Determine the Dynamics of Secondary Influenza Virus Infection and Explain Observed Viral Hierarchies

Influenza is an infectious disease that primarily attacks the respiratory system. Innate immunity provides both a very early defense to influenza virus invasion and an effective control of viral growth. Previous modelling studies of virus–innate immune response interactions have focused on infection with a single virus and, while improving our understanding of viral and immune dynamics, have been unable to effectively evaluate the relative feasibility of different hypothesised mechanisms of antiviral immunity. In recent experiments, we have applied consecutive exposures to different virus strains in a ferret model, and demonstrated that viruses differed in their ability to induce a state of temporary immunity or viral interference capable of modifying the infection kinetics of the subsequent exposure. These results imply that virus-induced early immune responses may be responsible for the observed viral hierarchy. Here we introduce and analyse a family of within-host models of re-infection viral kinetics which allow for different viruses to stimulate the innate immune response to different degrees. The proposed models differ in their hypothesised mechanisms of action of the non-specific innate immune response. We compare these alternative models in terms of their abilities to reproduce the re-exposure data. Our results show that 1) a model with viral control mediated solely by a virus-resistant state, as commonly considered in the literature, is not able to reproduce the observed viral hierarchy; 2) the synchronised and desynchronised behaviour of consecutive virus infections is highly dependent upon the interval between primary virus and challenge virus exposures and is consistent with virus-dependent stimulation of the innate immune response. Our study provides the first mechanistic explanation for the recently observed influenza viral hierarchies and demonstrates the importance of understanding the host response to multi-strain viral infections. Re-exposure experiments provide a new paradigm in which to study the immune response to influenza and its role in viral control.     

Proteomic analysis of the lungs of mice infected with different pathotypes of H5N1 avian influenza viruses

The virulence of influenza virus is determined by viral and host factors. Data on the genetic basis of the virulence of H5N1 influenza viruses have increased over the past decade; however, the contributions of host factors to the outcomes of H5N1 infection remain largely unknown. Here, we tested two chicken H5N1 viruses in mice and found that A/chicken/VN1214/2007 was nonlethal in mice and only replicated in the lung, whereas A/chicken/VN1180/2006 was highly lethal and replicated systemically in mice. To investigate the host response against these two different virus infections, we performed proteomic analysis by using 2D DIGE on the lung tissues of mice collected on days 1 and 3 postinoculation with different viruses or PBS as a control. Thirty-nine differentially expressed (DE) proteins related to "immune and stimulus response," "macromolecular biosynthesis and metabolism," and "cellular component and cytoskeleton" were identified in the virus-inoculated groups. Moreover, 13 DE proteins were identified between the two virus-inoculated groups, implying that these proteins may play important roles in the different outcomes of infection with these two viruses. Our data provide important information regarding the host response to mild and lethal H5N1 influenza virus infection.     

Exploring cell tropism as a possible contributor to influenza infection severity

Several mechanisms have been proposed to account for the marked increase in severity of human infections with avian compared to human influenza strains, including increased cytokine expression, poor immune response, and differences in target cell receptor affinity. Here, the potential effect of target cell tropism on disease severity is studied using a mathematical model for in-host influenza viral infection in a cell population consisting of two different cell types. The two cell types differ only in their susceptibility to infection and rate of virus production. We show the existence of a parameter regime which is characterized by high viral loads sustained long after the onset of infection. This finding suggests that differences in cell tropism between influenza strains could be sufficient to cause significant differences in viral titer profiles, similar to those observed in infections with certain strains of influenza A virus. The two target cell mathematical model offers good agreement with experimental data from severe influenza infections, as does the usual, single target cell model albeit with biologically unrealistic parameters. Both models predict that while neuraminidase inhibitors and adamantanes are only effective when administered early to treat an uncomplicated seasonal infection, they can be effective against more severe influenza infections even when administered late.