Effect of on-going and persistent infection on acute respiratory infection with influenza A

Humans are subject to infection with a wide range of commensal and pathogenic organisms. Each pathogen requires an appropriate immune response to eliminate or control the invading organism and minimise pathology. Many pathogens have evolved strategies to subvert or manipulate the immune response and establish on-going infections. Similarly acute respiratory infection with virulent strains of influenza A virus are often poorly controlled by the immune system and can cause severe immunopathology and even fatality as a result of an inappropriate and excessive inflammatory response called a ‘cytokine storm’. Morbidity due to influenza infection and exacerbation by the immune response can vary greatly between individuals. The effect of underlying infection on the immune system could contribute to the variation in response. The aim of this project was therefore to determine if co-infection with two pathogens that establish on-going infections could alter the immune response to influenza A and impact the outcome of infection. Persistent infections with filarial helminths can cause debilitating disease and significantly impact the immune response toward a skewed TH2 or regulatory phenotype in order to control pathology. In contrast, infection with gammaherpesviruses in an immunocompetent host causes an initial inflammatory ‘anti-viral’ response before becoming an asymptomatic, latent infection. In an immunocompromised host, gammaherpesviruses can reactivate and lead to clinical presentation of disease. This suggests that these viruses require an on-going immune response to control all stages of infection. Both filarial helminths and gammaherpesviruses are common infections in human populations and therefore mouse models of these infections provide relevant systems to study their potential role in influenza virus infections. In a BALB/c murine co-infection model, latent infection with the rodent gammaherpesvirus MHV-68 led to significantly decreased weight loss and clinical signs following high dose infection with A/WSN/33, (a H1N1 influenza A virus). This was coupled with decreased immunopathology in the lung and fewer infiltrating lymphocytes in the alveolar spaces and around larger airways, although infectious virus titres were not significantly reduced. This response was coupled with a decreased production of inflammatory cytokines and chemokines in co-infected mice 6 days post infection which correlated with the amelioration of pathogenesis in these animals. A repeat of the study in 129Sv/Ev IFNγR knock out mice showed the same protective effect in the co-infected mice, suggesting IFNγ is not critical for the protective phenotype. Mice infected with latent MHV-68 alone showed a significant increase in expression of T cell chemokines in the lung and alveolar macrophages had a significantly increased production of suppressor of cytokine signalling (SOCS-1) suggesting latent MHV-68 infection may impact the phenotype of macrophages in the lung, modulating the response to influenza co-infection. A co-infection model with a persistent rodent filarial helminth, Litomosoides sigmodontis and A/WSN/33 was also established in BALB/c mice. The L4 developmental stage of L. sigmodontis infection had no impact on co-infection with A/WSN/33. Adult stage worms, however, appeared to have a protective effect against A/WSN/33 pathogenesis. Co-infected mice had significantly delayed weight loss and clinical signs 3-5 days post infection. CD4+ and CD8+ T cells in the lung draining lymph nodes had significantly reduced TH1 and TH2 phenotypes (measured by cytokine production) compared with singly infected controls. IFNγ secreting CD4+ T cells in the lungs of co-infected mice also secreted increased levels of IL-10, suggesting an increase in regulation of the inflammatory response to A/WSN/33. At the full patent stage of L. sigmodontis infection, co-infection with A/WSN/33 led to increased clinical signs and significantly exacerbated weight loss. CD4+ and CD8+ T cells in the lung draining lymph nodes were inflammatory in L. sigmodontis infected mice alone as well as co-infected mice and there were no differences in the percentage of CD4+ T cells in the lung secreting IL-10 and IFNγ between co-infected and influenza infected mice. A loss in regulatory responses during the patent stage of L. sigmodontis infection may therefore contribute to the loss of protection against A/WSN/33 at this time point within the co-infection model. Understanding the impact of an underlying infection on the immune system could provide immune mechanisms that could be exploited to increase vaccine efficacy against influenza and similarly help to provide better treatment for individuals infected with influenza A. These results may also help predict the outcome of influenza A infection in individuals already infected with highly immunogenic, on-going infections

Macrophages from gut-corrected CF mice express human CFTR and lack a pro-inflammatory phenotype

Macrophages represent prominent immune orchestrators of cystic fibrosis (CF) inflammation and, as such, are an ever-increasing focus of CF research with several reports of intrinsic immune dysfunction related to loss of CFTR activity in macrophages themselves. Animal models of CF have contributed, in no small part, to a deepening of our understanding of the pathophysiology of the disease and towards therapeutic development. A commonly-used animal model in CF research is the Cftr(tm1Unc) Tg(FABP-hCFTR) mouse, which displays gut-specific expression of a human CFTR transgene in order to rescue the high rate of early mortality in Cftr-null mice associated with severe intestinal obstruction. We find significant variation in the response to inflammatory challenge of patient macrophages and cells derived from the Cftr(tm1Unc) Tg(FABP-hCFTR) mouse and show that macrophages derived from this mouse exhibit aberrant expression of human CFTR. This may contribute to the absence of inflammatory changes in this model

The Outcome of Neutrophil-T Cell Contact Differs Depending on Activation Status of Both Cell Types

Neutrophils and T cells exist in close proximity in lymph nodes and inflamed tissues duringhealth and disease. They are able to form stable interactions, with profound effects on thephenotype and function of the T cells. However, the outcome of these effects arefrequently contradictory; in some systems neutrophils suppress T cell proliferation, inothers they are activatory or present antigen directly. Published protocols modelling theseinteractions in vitro do not reflect the full range of interactions found in vivo; they do notexamine how activated and naïve T cells differentially respond to neutrophils, or whetherde-granulating or resting neutrophils induce different outcomes. Here, we established aculture protocol to ask these questions with human T cells and autologous neutrophils.We find that resting neutrophils suppress T cell proliferation, activation and cytokineproduction but that de-granulating neutrophils do not, and neutrophil-releasedintracellular contents enhance proliferation. Strikingly, we also demonstrate that T cellsearly in the activation process are susceptible to suppression by neutrophils, while laterstage T cells are not, and naïve T cells do not respond at all. Our protocol therefore allowsnuanced analysis of the outcome of interaction of these cells and may explain thecontradictory results observed previously

Concurrent infection with the filarial helminth Litomosoides sigmodontis attenuates or worsens Influenza A virus pathogenesis in a stage-dependent manner

Filarial helminths infect approximately 120 million people worldwide initiating a type 2 immune response in the host. Influenza A viruses stimulate a virulent type 1 pro-inflammatory immune response that in some individuals can cause uncontrolled immunopathology and fatality. Although coinfection with filariasis and influenza is a common occurrence, the impact of filarial infection on respiratory viral infection is unknown. The aim of this study was to determine the impact of pre-existing filarial infection on concurrent infection with influenza A virus. A murine model of co-infection was established using the filarial helminth Litomosoides sigmodontis and the H1N1 (A/WSN/33) influenza A virus (IAV). Co-infection was performed at 3 different stages of L. sigmodontis infection (larval, juvenile adult, and patency), and the impact of co-infection was determined by IAV induced weight loss and clinical signs, quantification of viral titres, and helminth counts. Significant alterations of IAV pathogenesis, dependent upon stage of infection, was observed on co-infection with L. sigmodontis. Larval stage L. sigmodontis infection alleviated clinical signs of IAV co-infection, whilst more established juvenile adult infection also significantly delayed weight loss. Viral titres remained unaltered at either infection stage. In contrast, patent L. sigmdodontis infection led to a reversal of age-related resistance to IAV infection, significantly increasing weight loss and clinical signs of infection as well as increasing IAV titre. These data demonstrate that the progression of influenza infection can be ameliorated or worsened by pre-existing filarial infection, with the outcome dependent upon the stage of filarial infection

SERS Microsensors for the Study of pH Regulation in Cystic Fibrosis Patient-Derived Airway Cultures

Acidification of the airway surface liquid in the respiratory system could play a role in the pathology of Cystic Fibrosis, but its low volume and proximity to the airway epithelium make it a challenging biological environment in which to noninvasively collect pH measurements. To address this challenge, we explored surface enhanced Raman scattering microsensors (SERS-MS), with a 4-mercaptobenzoic acid (MBA) pH reporter molecule, as pH sensors for the airway surface liquid of patient-derived in vitro models of the human airway. Using air–liquid interface (ALI) cultures to model the respiratory epithelium, we show that SERS-MS facilitates the optical measurement of trans-epithelial pH gradients between the airway surface liquid and the basolateral culture medium. SERS-MS also enabled the successful quantification of pH changes in the airway surface liquid following stimulation of the Cystic Fibrosis transmembrane conductance regulator (CFTR, the apical ion channel that is dysfunctional in Cystic Fibrosis airways). Finally, the influence of CFTR mutations on baseline airway surface liquid pH was explored by using SERS-MS to measure the pH in ALIs grown from Cystic Fibrosis and non-Cystic Fibrosis donors

High purity isolation of low density neutrophils casts doubt on their exceptionality in health and disease

Low density neutrophils (LDNs) are described in a number of inflammatory conditions, cancers and infections and associated with immunopathology, and a mechanistic role in disease. The role of LDNs at homeostasis in healthy individuals has not been investigated. We have developed an isolation protocol that generates high purity LDNs from healthy donors. Healthy LDNs were identical to healthy normal density neutrophils (NDNs), aside from reduced neutrophil extracellular trap formation. CD66b, CD16, CD15, CD10, CD54, CD62L, CXCR2, CD47 and CD11b were expressed at equivalent levels in healthy LDNs and NDNs and underwent apoptosis and ROS production interchangeably. Healthy LDNs had no differential effect on CD4(+) or CD8(+) T cell proliferation or IFNγ production compared with NDNs. LDNs were generated from healthy NDNs in vitro by activation with TNF, LPS or fMLF, suggesting a mechanism of LDN generation in disease however, we show neutrophilia in people with Cystic Fibrosis (CF) was not due to increased LDNs. LDNs are present in the neutrophil pool at homeostasis and have limited functional differences to NDNs. We conclude that increased LDN numbers in disease reflect the specific pathology or inflammatory environment and that neutrophil density alone is inadequate to classify discrete functional populations of neutrophils

The neutrophil antimicrobial peptide cathelicidin promotes Th17 differentiation

The host defence peptide cathelicidin (LL-37 in humans, mCRAMP in mice) is released from neutrophils by de-granulation, NETosis and necrotic death; it has potent anti-pathogen activity as well as being a broad immunomodulator. Here we report that cathelicidin is a powerful Th17 potentiator which enhances aryl hydrocarbon receptor (AHR) and RORγt expression, in a TGF-β1-dependent manner. In the presence of TGF-β1, cathelicidin enhanced SMAD2/3 and STAT3 phosphorylation, and profoundly suppressed IL-2 and T-bet, directing T cells away from Th1 and into a Th17 phenotype. Strikingly, Th17 but not Th1 cells were protected from apoptosis by cathelicidin. We show that cathelicidin is released by neutrophils in mouse lymph nodes and that cathelicidin-deficient mice display suppressed Th17 responses during inflammation, but not at steady state. We propose that the neutrophil cathelicidin is required for maximal Th17 differentiation, and that this is one method by which early neutrophilia directs subsequent adaptive immune responses