Our work is focused on understanding how immune responses in the lungs impact on the mechanisms of injury and repair. We are particularly interested in the role of invariant natural killer T cells (iNKT) and monocytes in immune-regulation and disease pathogenesis.

We have four areas of research that span cellular mechanisms to translational medicine:

• Understanding cellular and molecular interaction of iNKT cells with monocytes
• Examining the contribution of CD1d expression on lung epithelium to mucosal immunity in the lungs
• Examining the contribution of subsets of monocytes to lung injury and resolution of repair
• Using transcriptomics to develop molecular biomarker for predicting outcome in inflammatory lung disease and as a platform for discovery of mechanisms involved in lung immunepathology

These areas of immunology research cross-cut with our focus on the human diseases of idiopathic pulmonary fibrosis, sarcoidosis and severe influenza infection.

Crawshaw A, Kendrick YR, McMichael AJ, Ho LP. 2014. Abnormalities in iNKT cells are associated with impaired ability of monocytes to produce IL-10 and suppress T-cell proliferation in sarcoidosis. Eur J Immunol, 44 (7), pp. 2165-2174. Read abstract | Read more

Sarcoidosis is a multisystem granulomatous disorder characterized by marked T-cell expansion of T helper 1 (Th1) cells. The cause of T-cell overactivity is unknown. We hypothesized that interleukin-10 (IL-10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T-cell activity. Focusing on IL-10-producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid-naïve sarcoidosis patients (n = 51) produced less IL-10 compared to controls, and were less able to suppress T-cell proliferation. In addition, monocytic IL-10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT-specific defects might be responsible for this reduced IL-10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL-10 production. Moreover, iNKT cells enhanced monocytic IL-10 production in vitro. Defective IL-10 production and T-cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d- and cell contact-dependent process. We suggest that reduced iNKT-cell numbers in sarcoidosis may lead to impaired monocytic IL-10 production and unchecked T-cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes. Hide abstract

Cole SL, Benam KH, McMichael AJ, Ho LP. 2014. Involvement of the 4-1BB/4-1BBL pathway in control of monocyte numbers by invariant NKT cells. J Immunol, 192 (8), pp. 3898-3907. Read abstract | Read more

4-1BB is expressed on invariant (i)NKT cells, but its role is unclear. We showed previously that iNKT cells are involved in control of monocyte numbers during influenza A virus (IAV) infection and now question the role of the 4-1BB costimulatory pathway in the cross-talk between these cells. We found that iNKT cells and monocytes interact to promote expression of 4-1BB and 4-1BBL, respectively. Blockade of 4-1BB/L pathway under resting coculture conditions increased apoptosis of iNKT cells and monocytes. However, activation of iNKT cells overrides this survival signal, causing marked apoptosis of monocytes independent of 4-1BB/L. Blocking 4-1BBL in alpha-galactosylceramide-activated iNKT-monocyte cocultures reduced iNKT proliferation and abrogated monocytic IL-12 production. In vivo, expression of 4-1BB and 4-1BBL is increased on iNKT cells and Ly6C(hi) monocytes, respectively, during IAV infection, and there were lower frequencies of apoptosing Ly6C(hi) monocytes in the blood of iNKT knockout mice and higher numbers of monocytes in lungs compared with infected wild-type mice. Adoptive transfer of iNKT cells into the lungs of these mice reduced lung Ly6C(hi) monocytes levels, even when iNKT cells were preincubated with 4-1BB blocking Abs. These findings suggest that under resting conditions, 4-1BB/L engagement during iNKT-monocyte interaction promotes survival of these cells. When iNKT cells are activated, whether by alpha-galactosylceramide or during IAV infection, iNKT cells induced apoptosis of monocytes via a 4-1BB/L-independent mechanism, reducing monocyte numbers. 4-1BB/L costimulation amplified monocyte-mediated proliferation of iNKT cells, indirectly providing a method for monocytes to control their own numbers during infection. Hide abstract

Bloom CI, Graham CM, Berry MP, Rozakeas F, Redford PS, Wang Y, Xu Z, Wilkinson KA, Wilkinson RJ, Kendrick Y, Devouassoux G, Ferry T, Miyara M, Bouvry D, Valeyre D, Gorochov G, Blankenship D, Saadatian M, Vanhems P, Beynon H, Vancheeswaran R, Wickremasinghe M, Chaussabel D, Banchereau J, Pascual V, Ho LP, Lipman M, O'Garra A et al. 2013. Transcriptional blood signatures distinguish pulmonary tuberculosis, pulmonary sarcoidosis, pneumonias and lung cancers. PLoS One, 8 (8), pp. e70630. Read abstract | Read more

RATIONALE: New approaches to define factors underlying the immunopathogenesis of pulmonary diseases including sarcoidosis and tuberculosis are needed to develop new treatments and biomarkers. Comparing the blood transcriptional response of tuberculosis to other similar pulmonary diseases will advance knowledge of disease pathways and help distinguish diseases with similar clinical presentations. OBJECTIVES: To determine the factors underlying the immunopathogenesis of the granulomatous diseases, sarcoidosis and tuberculosis, by comparing the blood transcriptional responses in these and other pulmonary diseases. METHODS: We compared whole blood genome-wide transcriptional profiles in pulmonary sarcoidosis, pulmonary tuberculosis, to community acquired pneumonia and primary lung cancer and healthy controls, before and after treatment, and in purified leucocyte populations. MEASUREMENTS AND MAIN RESULTS: An Interferon-inducible neutrophil-driven blood transcriptional signature was present in both sarcoidosis and tuberculosis, with a higher abundance and expression in tuberculosis. Heterogeneity of the sarcoidosis signature correlated significantly with disease activity. Transcriptional profiles in pneumonia and lung cancer revealed an over-abundance of inflammatory transcripts. After successful treatment the transcriptional activity in tuberculosis and pneumonia patients was significantly reduced. However the glucocorticoid-responsive sarcoidosis patients showed a significant increase in transcriptional activity. 144-blood transcripts were able to distinguish tuberculosis from other lung diseases and controls. CONCLUSIONS: Tuberculosis and sarcoidosis revealed similar blood transcriptional profiles, dominated by interferon-inducible transcripts, while pneumonia and lung cancer showed distinct signatures, dominated by inflammatory genes. There were also significant differences between tuberculosis and sarcoidosis in the degree of their transcriptional activity, the heterogeneity of their profiles and their transcriptional response to treatment. Hide abstract

Zhang YH, Zhao Y, Li N, Peng YC, Giannoulatou E, Jin RH, Yan HP, Wu H, Liu JH, Liu N, Wang DY, Shu YL, Ho LP, Kellam P, McMichael A, Dong T et al. 2013. Interferon-induced transmembrane protein-3 genetic variant rs12252-C is associated with severe influenza in Chinese individuals. Nat Commun, 4 pp. 1418. Read abstract | Read more

The SNP rs12252-C allele alters the function of interferon-induced transmembrane protein-3 increasing the disease severity of influenza virus infection in Caucasians, but the allele is rare. However, rs12252-C is much more common in Han Chinese. Here we report that the CC genotype is found in 69% of Chinese patients with severe pandemic influenza A H1N1/09 virus infection compared with 25% in those with mild infection. Specifically, the CC genotype was estimated to confer a sixfold greater risk for severe infection than the CT and TT genotypes. More importantly, because the risk genotype occurs with such a high frequency, its effect translates to a large population-attributable risk of 54.3% for severe infection in the Chinese population studied compared with 5.4% in Northern Europeans. Interferon-induced transmembrane protein-3 genetic variants could, therefore, have a strong effect of the epidemiology of influenza in China and in people of Chinese descent. Hide abstract

Martinez FO, Helming L, Milde R, Varin A, Melgert BN, Draijer C, Thomas B, Fabbri M, Crawshaw A, Ho LP, Ten Hacken NH, Cobos Jiménez V, Kootstra NA, Hamann J, Greaves DR, Locati M, Mantovani A, Gordon S et al. 2013. Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences. Blood, 121 (9), pp. e57-e69. Read abstract | Read more

The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4–activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma. Hide abstract

Zhao Y, Zhang YH, Denney L, Young D, Powell TJ, Peng YC, Li N, Yan HP, Wang DY, Shu YL, Kendrick Y, McMichael AJ, Ho LP, Dong T et al. 2012. High levels of virus-specific CD4+ T cells predict severe pandemic influenza A virus infection. Am J Respir Crit Care Med, 186 (12), pp. 1292-1297. Read abstract | Read more

RATIONALE: T-cell responses have been implicated in control and exacerbation of lung injury during influenza A virus (IAV) infection. OBJECTIVES: To examine the breadth and magnitude of influenza-specific CD4(+) and CD8(+) T-cell responses during acute phase of infection. METHODS: Influenza-specific T-cell response to the entire pandemic H1N1/09 IAV proteome and T cell-related cytokine levels were measured in blood from previously healthy individuals with mild (n = 32) and severe (n = 16) IAV infection during the 2009 influenza pandemic. Virus-specific T-cell response in lung and blood was also performed in two acutely infected, severely ill patients using fluorescent-conjugated pdmH1N1/09 Matrix-MHC-I tetrameric complexes. MEASUREMENTS AND MAIN RESULTS: Strong and broad CD4(+) but not CD8(+) T-cell responses were observed in the blood, and were higher in those with severe disease. Antigen-specific CD8(+) T cells in the lungs were on average 45-fold higher compared with blood in severely ill patients. Paradoxically, in patients with severe disease, IL-17, IL-2, IL-4, and IFN-γ levels were significantly decreased. CONCLUSIONS: High levels of circulating virus-specific CD4(+) T cells to two viral internal proteins (nucleoprotein and matrix) in the first phase of infection are associated with subsequent development of severe IAV infection. This finding could be an early and specific marker for ensuing clinical deterioration. Contrasting levels of antigen-specific CD8(+) T cells in lungs and blood have implications on design and analysis of clinical trials for T-cell vaccines because measurements of T cells in the periphery may not reflect events in the lungs. Hide abstract

Wegmann F, Gartlan KH, Harandi AM, Brinckmann SA, Coccia M, Hillson WR, Kok WL, Cole S, Ho LP, Lambe T, Puthia M, Svanborg C, Scherer EM, Krashias G, Williams A, Blattman JN, Greenberg PD, Flavell RA, Moghaddam AE, Sheppard NC, Sattentau QJ et al. 2012. Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens. Nat Biotechnol, 30 (9), pp. 883-888. Read abstract | Read more

Protection against mucosally transmitted infections probably requires immunity at the site of pathogen entry, yet there are no mucosal adjuvant formulations licensed for human use. Polyethyleneimine (PEI) represents a family of organic polycations used as nucleic acid transfection reagents in vitro and DNA vaccine delivery vehicles in vivo. Here we show that diverse PEI forms have potent mucosal adjuvant activity for viral subunit glycoprotein antigens. A single intranasal administration of influenza hemagglutinin or herpes simplex virus type-2 (HSV-2) glycoprotein D with PEI elicited robust antibody-mediated protection from an otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen, which were taken up by antigen-presenting cells in vitro and in vivo, promoted dendritic cell trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host double-stranded DNA that triggered Irf3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use. Hide abstract

Denney L, Kok WL, Cole SL, Sanderson S, McMichael AJ, Ho LP. 2012. Activation of invariant NKT cells in early phase of experimental autoimmune encephalomyelitis results in differentiation of Ly6Chi inflammatory monocyte to M2 macrophages and improved outcome. J Immunol, 189 (2), pp. 551-557. Read abstract | Read more

Neuropathology in multiple sclerosis is closely linked to presence of macrophages in the CNS. Both M1 (inflammatory) and M2 (alternatively activated, noninflammatory) macrophages are found in the inflamed CNS and thought to differentiate from infiltrating monocytes. It is unclear whether the balance of M1 and M2 macrophages can be altered and whether this affects disease outcome. We show in this article that Ly6C(hi) inflammatory monocytes are the early and dominant infiltrating cells in the CNS during experimental autoimmune encephalomyelitis, a model for the acute phase of multiple sclerosis. Activation of invariant NKT (iNKT) cells reduced the frequency of Ly6C(hi) monocytes and increased the proportion of M2 macrophages in the CNS with associated improvement in neurologic impairment. In contrast, iNKT-deficient mice showed higher numbers of Ly6C(hi) monocytes, reduced M2, and much more severe disease. Adoptive transfer of M2-enriched cells to iNKT-deficient mice markedly improved neurologic impairment. In vitro and in vivo experiments showed that iNKT cells promote differentiation of monocytes to M2 macrophages in an IL-4 and CD1d-dependent process. These findings indicate that infiltrating Ly6C(hi) inflammatory monocytes are early players in acute neuroinflammation and that their frequency and differentiation can be influenced by activation of iNKT cells with resultant improvement in disease outcome. Hide abstract

Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, Chin CR, Feeley EM, Sims JS, Adams DJ, Wise HM, Kane L, Goulding D, Digard P, Anttila V, Baillie JK, Walsh TS, Hume DA, Palotie A, Xue Y, Colonna V, Tyler-Smith C, Dunning J, Gordon SB, Everingham K, Dawson H, Hope D, Ramsay P, Walsh Local Lead Investigator TS, Campbell A, Kerr S, Harrison D, Rowan K, Addison J, Donald N, Galt S, Noble D, Taylor J, Webster Local Lead Investigator N, Taylor Local Lead Investigator I, Aldridge Local Lead Investigator J, Dornan R, Richard C, Gilmour D, Simmons Local Lead Investigator R, White Local Lead Investigator R, Jardine C, Williams Local Lead Investigator D, Booth Local Lead Investigator M, Quasim T, Watson V, Henry P, Munro F, Bell L, Ruddy Local Lead Investigator J, Cole Local Lead Investigator S, Southward J, Allcoat P, Gray S, McDougall Local Lead Investigator M, Matheson J, Whiteside Local Lead Investigator J, Alcorn D, Rooney Local Lead Investigator K, Sundaram R, Imrie Local Lead Investigator G, Bruce J, McGuigan K, Moultrie Local Lead Investigator S, Cairns Local Lead Investigator C, Grant J, Hughes M, Murdoch Local Lead Investigator C, Davidson Local Lead Investigator A, Harris G, Paterson R, Wallis Local Lead Investigator C, Binning Local Lead Investigator S, Pollock M, Antonelli J, Duncan A, Gibson J, McCulloch C, Murphy L, Haley C, Faulkner G, Freeman T, Hume DA, Baillie Principal Investigator JK, Chaussabel D, Adamson WE, Carman WF, Thompson C, Zambon MC, Aylin P, Ashby D, Barclay WS, Brett SJ, Cookson WO, Drumright LN, Dunning J, Elderfield RA, Garcia-Alvarez L, Gazzard BG, Griffiths MJ, Habibi MS, Hansel TT, Herberg JA, Holmes AH, Hussell T, Johnston SL, Kon OM, Levin M, Moffatt MF, Nadel S, Openshaw PJ, Warner JO, Aston SJ, Gordon SB, Hay A, McCauley J, O'Garra A, Banchereau J, Hayward A, Kellam P, Baillie JK, Hume DA, Simmonds P, McNamara PS, Semple MG, Smyth RL, Nguyen-Van-Tam JS, Ho L-P, McMichael AJ, Kellam P, Smyth RL, Openshaw PJ, Dougan G, Brass AL, Kellam P et al. 2012. IFITM3 restricts the morbidity and mortality associated with influenza Nature,

Kok WL, Denney L, Benam K, Cole S, Clelland C, McMichael AJ, Ho LP. 2012. Pivotal Advance: Invariant NKT cells reduce accumulation of inflammatory monocytes in the lungs and decrease immune-pathology during severe influenza A virus infection. J Leukoc Biol, 91 (3), pp. 357-368. Read abstract | Read more

Little is known of how a strong immune response in the lungs is regulated to minimize tissue injury during severe influenza A virus (IAV) infection. Here, using a model of lethal, high-pathogenicity IAV infection, we first show that Ly6C(hi)Ly6G(-) inflammatory monocytes, and not neutrophils, are the main infiltrate in lungs of WT mice. Mice devoid of iNKT cells (Jα18(-/-) mice) have increased levels of inflammatory monocytes, which correlated with increased lung injury and mortality (but not viral load). Activation of iNKT cells correlated with reduction of MCP-1 levels and improved outcome. iNKT cells were able to selectively lyse infected, MCP-1-producing monocytes in vitro, in a CD1d-dependent process. Our study provides a detailed profile and kinetics of innate immune cells in the lungs during severe IAV infection, highlighting inflammatory monocytes as the major infiltrate and identifying a role for iNKT cells in control of these cells and lung immune-pathology. Hide abstract

Benam KH, Kok W, McMichael AJ, Ho LP. 2011. Alternative spliced CD1d transcripts in human bronchial epithelial cells PLoS ONE, 6 (8), Read abstract | Read more

CD1d is a MHC I like molecule which presents glycolipid to natural killer T (NKT) cells, a group of cells with diverse but critical immune regulatory functions in the immune system. These cells are required for optimal defence against bacterial, viral, protozoan, and fungal infections, and control of immune-pathology and autoimmune diseases. CD1d is expressed on antigen presenting cells but also found on some non-haematopoietic cells. However, it has not been observed on bronchial epithelium, a site of active host defence in the lungs. Here, we identify for the first time, CD1D mRNA variants and CD1d protein expression on human bronchial epithelial cells, describe six alternatively spliced transcripts of this gene in these cells; and show that these variants are specific to epithelial cells. These findings provide the basis for investigations into a role for CD1d in lung mucosal immunity. © 2011 Benam et al. Hide abstract

Lockstone HE, Sanderson S, Kulakova N, Baban D, Leonard A, Kok WL, McGowan S, McMichael AJ, Ho LP. 2010. Gene set analysis of lung samples provides insight into pathogenesis of progressive, fibrotic pulmonary sarcoidosis. Am J Respir Crit Care Med, 181 (12), pp. 1367-1375. Read abstract | Read more

RATIONALE: Approximately 60 to 70% of patients with pulmonary sarcoidosis have disease that resolves spontaneously; the rest follow a chronic course with varying levels of fibrosis. It is unclear why some patients progress and if treatment affects outcome. OBJECTIVES: To determine differential gene expression profile in lungs of patients with self-limiting sarcoidosis compared to those with progressive-fibrotic disease, and to analyze the biological relevance of these differentially expressed genes. METHODS: We examined microarray expression of 26,626 genes in transbronchial biopsies of granulomatous areas in lungs of patients with active but self-limiting (n = 8) versus those with active, progressive (+/- fibrotic) pulmonary disease (n = 7). MEASUREMENTS AND MAIN RESULTS: Three hundred thirty-four genes were differentially expressed between the two groups (P < 0.01, Bayesian moderated t test). Gene Set Enrichment Analysis showed over-representation of gene-sets (defined by Gene Ontology) related to host immune activation, proliferation, and defense, among genes up-regulated in the progressive-fibrotic group (FDR q < 0.0001 for the top 43 gene sets), and a marked enrichment of, and similarity in gene expression profiles between, progressive-fibrotic sarcoidosis and hypersensitivity pneumonitis (HP), (q < 0.001), but not idiopathic pulmonary fibrosis (IPF). CONCLUSIONS: The findings suggest that patients with progressive/fibrotic pulmonary sarcoidosis have intense immune activity related to host defense in their lungs, with processes more similar to HP than IPF. The study also demonstrates that transbronchial lung biopsy samples can provide good-quality RNA for gene expression profiling, supporting its potential use as a prognostic classifier for pulmonary sarcoidosis. Hide abstract

Denney L, Aitken C, Li CK, Wilson-Davies E, Kok WL, Clelland C, Rooney K, Young D, Dong T, McMichael AJ, Carman WF, Ho LP et al. 2010. Reduction of natural killer but not effector CD8 T lymphocytes in three consecutive cases of severe/lethal H1N1/09 influenza A virus infection. PLoS One, 5 (5), pp. e10675. Read abstract | Read more

BACKGROUND: The cause of severe disease in some patients infected with pandemic influenza A virus is unclear. METHODOLOGY/PRINCIPAL FINDINGS: We present the cellular immunology profile in the blood, and detailed clinical (and post-mortem) findings of three patients with rapidly progressive infection, including a pregnant patient who died. The striking finding is of reduction in natural killer (NK) cells but preservation of activated effector CD8 T lymphocytes; with viraemia in the patient who had no NK cells. Comparison with control groups suggests that the reduction of NK cells is unique to these severely ill patients. CONCLUSION/SIGNIFICANCE: Our report shows markedly reduced NK cells in the three patients that we sampled and raises the hypothesis that NK may have a more significant role than T lymphocytes in controlling viral burden when the host is confronted with a new influenza A virus subtype. Hide abstract

Ho LP, Denney L, Luhn K, Teoh D, Clelland C, McMichael AJ. 2008. Activation of invariant NKT cells enhances the innate immune response and improves the disease course in influenza A virus infection. Eur J Immunol, 38 (7), pp. 1913-1922. Read abstract | Read more

Invariant NKT (iNKT) cells have an indubitable role in antiviral immunity, although the mechanisms by which these cells exert their functions are not fully elucidated. With the emerging importance of high-pathogenicity influenza A virus infections in humans, we questioned whether iNKT cells contribute to immune defence against influenza A virus and whether activation of these cells influences outcome. We show that activation of iNKT cells with alpha-galactosylceramide (alpha-GC) during influenza virus infection transiently enhanced early innate immune response without affecting T cell immunity, and reduced early viral titres in lungs of C57BL/6 mice. This is accompanied by a better disease course with improved weight loss profile. Temporal changes in iNKT cells in the liver, blood and lungs suggest activation and migration of iNKT cells from the liver to the lungs in mice that were administered alpha-GC. Improvement in viral titres appears dependent on activation of iNKT cells via the intraperitoneal route since intranasal administration of alpha-GC did not have the same effect. We conclude that activation of iNKT cells enhances early innate immune response in the lungs and contribute to antiviral immunity and improved disease course in influenza A virus infection. Hide abstract

Ho LP, Urban BC, Thickett DR, Davies RJ, McMichael AJ. 2005. Deficiency of a subset of T-cells with immunoregulatory properties in sarcoidosis. Lancet, 365 (9464), pp. 1062-1072. Read abstract | Read more

BACKGROUND: Sarcoidosis is a multisystem disorder that predominantly involves the lungs, characterised by a T-helper 1 (Th1) biased CD4-positive T-cell response and granuloma formation, for which the explanation is unknown. A newly identified subset of T-cells with immunoregulatory functions, CD1d-restricted natural-killer T (NKT) cells, has been shown to protect against disorders with increased CD4-positive Th1 responses in animals. We explored whether abnormalities in these cells are implicated in the pathogenesis of sarcoidosis. METHODS: We