The balance between protective and pathogenic immune responses in the TB-infected lung.

Tuberculosis is a disease of the lung, and efficient transmission is dependent on the generation of a lesion in the lung, which results in a bacterium-laden cough. Mycobacterium tuberculosis (Mtb) is able to manipulate both the innate and acquired immune response of the host. This manipulation results in an effective CD4(+) T cell response that limits disease throughout the body but can also promote the development of progressively destructive lesions in the lung. In this way Mtb infection can result in an ambulatory individual who has a lesion in the lung capable of transmitting Mtb. The inflammatory environment within the lung lesion is manipulated by Mtb throughout infection and can limit the expression of acquired immunity by a variety of pathways.

Effect of high intensity interval training and moderate intensity continuous training on lymphoid, myeloid and inflammatory cells in kidney transplant recipients.

Kidney transplantations are seen to be a double-edge sword. Transplantations help to partially restore renal function, however there are a number of health-related co-morbidities associated with transplantation. Cardiovascular disease (CVD), malignancy and infections all limit patient and graft survival. Immunosuppressive medications alter innate and adaptive immunity and can result in immune dysfunction. Over suppression of the immune system can result in infections whereas under suppression can result in graft rejection. Exercise is a known therapeutic intervention with many physiological benefits. Its effects on immune function are not well characterised and may include both positive and negative influences depending on the type, intensity, and duration of the exercise bout. High intensity interval training (HIIT) has become more popular due to it resulting in improvements to tradional and inflammatory markers of cardiovascular (CV) risk in clinical and non-clinical populations. Though these improvements are similar to those seen with moderate intensity exercise, HIIT requires a shorter overall time commitment, whilst improvements can also be seen even with a reduced exercise volume. The purpose of this study was to explore the physiolocial and immunological impact of 8-weeks of HIIT and moderate intensity continuous training (MICT) in kidney transplan recipients (KTRs). In addition, the natural variations of immune and inflammatory cells in KTRs and non-CKD controls over a longitudinal period are explored. Newly developed multi-colour flow cytometry methods were devised to identify and characterise immune cell populations. Twenty-six KTRs were randomised into one of two HIIT protocols or MICT: HIIT A (n=8; 4-, 2-, and 1-min intervals; 80-90% VO2peak), HIIT B (n=8, 4x4 min intervals; 80-90% VO2peak), or MICT (n=8, ~40 min; 50-60% VO2peak) for 24 supervised sessions on a stationary bike (approx. 3x/week over 8 ± 2 weeks). Blood samples taken pre-training, mid training, post-training and 3 months later. Novel multi-colour flow cytometric panels were developed to characterise lymphoid and myeloid cell population from peripheral blood mononuclear cells. No changes were observed for circulating immune and inflammatory cells over the 8-week interventions. The feasibility study does not suggest that exercise programmes using HIIT and MICT protocols elicit adverse negative effects on immunity in KTRs. Therefore, such protocols may be immunologically safe for these patients. The inability of the participants to achieve the target exercise intensities may be due to physiological abnormalities in this population which warrants further investigation.

CD25-Targeted IL-2 Signals Promote Improved Outcomes of Influenza Infection and Boost Memory CD4 T Cell Formation.

IL-2 is a pleotropic cytokine with potent pro- and anti-inflammatory effects. These divergent impacts can be directed in vivo by forming complexes of IL-2 and anti-IL-2 mAbs (IL-2C) to target IL-2 to distinct subsets of cells based on their expression of subunits of the IL-2R. In this study, we show that treatment of mice with a prototypical anti-inflammatory IL-2C, JES6-1-IL-2C, best known to induce CD25+ regulatory CD4 T cell expansion, surprisingly causes robust induction of a suite of inflammatory factors. However, treating mice infected with influenza A virus with this IL-2C reduces lung immunopathology. We compare the spectrum of inflammatory proteins upregulated by pro- and anti-inflammatory IL-2C treatment and uncover a pattern of expression that reveals potentially beneficial versus detrimental aspects of the influenza-associated cytokine storm. Moreover, we show that anti-inflammatory IL-2C can deliver survival signals to CD4 T cells responding to influenza A virus that improve their memory fitness, indicating a novel application of IL-2 to boost pathogen-specific T cell memory while simultaneously reducing immunopathology.

Memory CD4 T cell-derived IL-2 synergizes with viral infection to exacerbate lung inflammation.

Defining the most penetrating correlates of protective memory T cells is key for designing improved vaccines and T cell therapies. Here, we evaluate how interleukin (IL-2) production by memory CD4 T cells, a widely held indicator of their protective potential, impacts immune responses against murine influenza A virus (IAV). Unexpectedly, we show that IL-2-deficient memory CD4 T cells are more effective on a per cell basis at combating IAV than wild-type memory cells that produce IL-2. Improved outcomes orchestrated by IL-2-deficient cells include reduced weight loss and improved respiratory function that correlate with reduced levels of a broad array of inflammatory factors in the infected lung. Blocking CD70-CD27 signals to reduce CD4 T cell IL-2 production tempers the inflammation induced by wild-type memory CD4 T cells and improves the outcome of IAV infection in vaccinated mice. Finally, we show that IL-2 administration drives rapid and extremely potent lung inflammation involving NK cells, which can synergize with sublethal IAV infection to promote acute death. These results suggest that IL-2 production is not necessarily an indicator of protective CD4 T cells, and that the lung environment is particularly sensitive to IL-2-induced inflammation during viral infection.

Innate IFN-γ-Producing Cells Developing in the Absence of IL-2 Receptor Common γ-Chain.

IFN-γ is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-γ reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-γ-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-γ by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common γ-chain (γc) genes (Rag2-/-γc-/-), indicating their innate nature and their γc cytokine independence. Rag2-/-γc-/- mice are as resistant to Mycobacterium avium as Rag2-/- mice, whereas Rag2-/- mice lacking IFN-γ are more susceptible than either Rag2-/- or Rag2-/-γc-/- These lineage-negative CD45+/Thy1.2+ cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2-/-γc-/- animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-γ-producing cells. Accordingly, Thy1.2-specific mAb administration to infected Rag2-/-γc-/- animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2+ non-NK innate-like cells present in the liver expresses IFN-γ and can confer protection against M. avium infection in immunocompromised mice.

The onset of adaptive immunity in the mouse model of tuberculosis and the factors that compromise its expression.

Mycobacterium tuberculosis (Mtb) has been evolving with its human host for over 50 000 years and is an exquisite manipulator of the human immune response. It induces both a strong inflammatory and a strong acquired immune response, and Mtb then actively regulates these responses to create an infectious lesion in the lung while maintaining a relatively ambulatory host. The CD4(+) T cell plays a critical yet contradictory role in this process by both controlling disseminated disease while promoting the development of the lesion in the lung that mediates transmission. In light of this manipulative relationship between Mtb and the human immune response, it is not surprising that our ability to vaccinate against tuberculosis (TB) has not been totally successful. To overcome the current impasse in vaccine development, we need to define the phenotype of CD4(+) T cells that mediate protection and to determine those bacterial and host factors that regulate the effective function of these cells. In this review, we describe the initiation and expression of T cells during TB as well as the fulminant inflammatory response that can compromise T-cell function and survival.

TNFα and IFNγ but not perforin are critical for CD8 T cell-mediated protection against pulmonary Yersinia pestis infection.

Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE69-77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE69-77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE69-77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE69-77-mediated protection. In contrast, YopE69-77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias.

IL12Rß1ΔTM is a secreted product of il12rb1 that promotes control of extrapulmonary tuberculosis.

IL12RB1 is a human gene that is important for resistance to Mycobacterium tuberculosis infection. IL12RB1 is expressed by multiple leukocyte lineages, and encodes a type I transmembrane protein (IL12Rß1) that associates with IL12p40 and promotes the development of host-protective T(H)1 cells. Recently, we observed that il12rb1­the mouse homolog of IL12RB1­is alternatively spliced by leukocytes to produce a second isoform (IL12Rß1ΔTM) that has biological properties distinct from IL12Rß1. Although the expression of IL12Rß1ΔTM is elicited by M. tuberculosis in vivo, and its overexpression enhances IL12p40 responsiveness in vitro, the contribution of IL12Rß1ΔTM to controlling M. tuberculosis infection has not been tested. Here, we demonstrate that IL12Rß1ΔTM represents a secreted product of il12rb1 that, when absent from mice, compromises their ability to control M. tuberculosis infection in extrapulmonary organs. Furthermore, elevated M. tuberculosis burdens in IL12Rß1ΔTM-deficient animals are associated with decreased lymph node cellularity and a decline in TH1 development. Collectively, these data support a model wherein IL12Rß1ΔTM is a secreted product of il12rb1 that promotes resistance to M. tuberculosis infection by potentiating T(H) cells response to IL-12.

THEMIS is required for pathogenesis of cerebral malaria and protection against pulmonary tuberculosis.

We identify an N-ethyl-N-nitrosourea (ENU)-induced I23N mutation in the THEMIS protein that causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium berghei ANKA. Themis(I23N) homozygous mice show reduced CD4(+) and CD8(+) T lymphocyte numbers. ECM resistance in P. berghei ANKA-infected Themis(I23N) mice is associated with decreased cerebral cellular infiltration, retention of blood-brain barrier integrity, and reduced proinflammatory cytokine production. THEMIS(I23N) protein expression is absent from mutant mice, concurrent with the decreased THEMIS(I23N) stability observed in vitro. Biochemical studies in vitro and functional complementation in vivo in Themis(I23N/+):Lck(-/+) doubly heterozygous mice demonstrate that functional coupling of THEMIS to LCK tyrosine kinase is required for ECM pathogenesis. Damping of proinflammatory responses in Themis(I23N) mice causes susceptibility to pulmonary tuberculosis. Thus, THEMIS is required for the development and ultimately the function of proinflammatory T cells. Themis(I23N) mice can be used to study the newly discovered association of THEMIS (6p22.33) with inflammatory bowel disease and multiple sclerosis.

Cellular response to mycobacteria: balancing protection and pathology.

There has been a recent increase in our understanding of T cell responses during mycobacterial infection; however, we have not yet identified the protective mechanisms capable of mediating vaccine-induced protection in the lung. Novel approaches have allowed the determination of the kinetics and location of naïve T cell activation, as well as the factors that affect of antigen-specific T cell responses, and the balance between protective and immunopathological consequences during the chronic stages of infection. With an urgent need for new and more efficient vaccination strategies, the integration of these data will result in improved vaccine strategies.

Nitric oxide inhibits the accumulation of CD4+CD44hiTbet+CD69lo T cells in mycobacterial infection.

Animals lacking the inducible nitric oxide synthase gene (nos2(-/-)) are less susceptible to Mycobacterium avium strain 25291 and lack nitric oxide-mediated immunomodulation of CD4(+) T cells. Here we show that the absence of nos2 results in increased accumulation of neutrophils and both CD4(+) and CD8(+) T cells within the M. avium containing granuloma. Examination of the T-cell phenotype in M. avium infected mice demonstrated that CD4(+)CD44(hi) effector T cells expressing the Th1 transcriptional regulator T-bet (T-bet(+)) were specifically reduced by the presence of nitric oxide. Importantly, the T-bet(+) effector population could be separated into CD69(hi) and CD69(lo) populations, with the CD69(lo) population only able to accumulate during chronic infection within infected nos2(-/-) mice. Transcriptomic comparison between CD4(+)CD44(hi)CD69(hi) and CD4(+)CD44(hi)CD69(lo) populations revealed that CD4(+)CD44(hi)CD69(lo) cells had higher expression of the integrin itgb1/itga4 (VLA-4, CD49d/CD29). Inhibition of Nos2 activity allowed increased accumulation of the CD4(+) CD44(hi)T-bet(+)CD69(lo) population in WT mice as well as increased expression of VLA-4. These data support the hypothesis that effector T cells in mycobacterial granulomata are not a uniform effector population but exist in distinct subsets with differential susceptibility to the regulatory effects of nitric oxide.

IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung.

IL-23 is required for the IL-17 response to infection with Mycobacterium tuberculosis, but is not required for the early control of bacterial growth. However, mice deficient for the p19 component of IL-23 (Il23a(-/-)) exhibit increased bacterial growth late in infection that is temporally associated with smaller B cell follicles in the lungs. Cxcl13 is required for B cell follicle formation and immunity during tuberculosis. The absence of IL-23 results in decreased expression of Cxcl13 within M. tuberculosis-induced lymphocyte follicles in the lungs, and this deficiency was associated with increased cuffing of T cells around the vessels in the lungs of these mice. Il23a(-/-) mice also poorly expressed IL-17A and IL-22 mRNA. These cytokines were able to induce Cxcl13 in mouse primary lung fibroblasts, suggesting that these cytokines are likely involved in B cell follicle formation. Indeed, IL-17RA-deficient mice generated smaller B cell follicles early in the response, whereas IL-22-deficient mice had smaller B cell follicles at an intermediate time postinfection; however, only Il23a(-/-) mice had a sustained deficiency in B cell follicle formation and reduced immunity. We propose that in the absence of IL-23, expression of long-term immunity to tuberculosis is compromised due to reduced expression of Cxcl13 in B cell follicles and reduced ability of T cells to migrate from the vessels and into the lesion. Further, although IL-17 and IL-22 can both contribute to Cxcl13 production and B cell follicle formation, it is IL-23 that is critical in this regard.

Cytokines in the balance of protection and pathology during mycobacterial infections.

The outcome of natural infections with pathogenic mycobacteria can range from early asymptomatic clearance through latent infection to clinical disease. Different host and pathogen-specific factors have been implicated in determining the outcome of these infections; however, it is clear that the interaction of mycobacteria with the innate and acquired components of the immune system plays a central role. Specifically, the recognition of mycobacterial components by innate immune cells through different pathogen recognition receptors (PPRs) induces a cytokine response that can promote early control of the infection. In fact, in the majority of individuals that come into contact with mycobacteria, this response is enough to control the infection. Among PRRs, Toll-like receptors (TLRs), Nucleotide Oligomerization Domain (NOD)-like receptors, and C-type lectins have all been implicated in recognition of mycobacteria and in the initiation of the cytokine response. Defining the mechanisms by which distinct mycobacterial components and their receptors stimulate the immune response is an area of intense research.

Opposing biological functions of tryptophan catabolizing enzymes during intracellular infection.

Recent studies have underscored physiological and pathophysiological roles for the tryptophan-degrading enzyme indolamine 2,3-dioxygenase (IDO) in immune counterregulation. However, IDO was first recognized as an antimicrobial effector, restricting tryptophan availability to Toxoplasma gondii and other pathogens in vitro. The biological relevance of these findings came under question when infectious phenotypes were not forthcoming in IDO-deficient mice. The recent discovery of an IDO homolog, IDO-2, suggested that the issue deserved reexamination. IDO inhibition during murine toxoplasmosis led to 100% mortality, with increased parasite burdens and no evident effects on the immune response. Similar studies revealed a counterregulatory role for IDO during leishmaniasis (restraining effector immune responses and parasite clearance), and no evident role for IDO in herpes simplex virus type 1 (HSV-1) infection. Thus, IDO plays biologically important roles in the host response to diverse intracellular infections, but the dominant nature of this role--antimicrobial or immunoregulatory--is pathogen-specific.

Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice.

Introduction: The large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice. Methods: The investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice. Results: We found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs. Discussion: Having demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions.

Interleukin 12p40 is required for dendritic cell migration and T cell priming after Mycobacterium tuberculosis infection.

Migration of dendritic cells (DCs) to the draining lymph node (DLN) is required for the activation of naive T cells. We show here that migration of DCs from the lung to the DLN after Mycobacterium tuberculosis (Mtb) exposure is defective in mice lacking interleukin (IL)-12p40. This defect compromises the ability of IL-12p40-deficient DCs to activate naive T cells in vivo; however, DCs that express IL-12p40 alone can activate naive T cells. Treatment of IL-12p40-deficient DCs with IL-12p40 homodimer (IL-12(p40)(2)) restores Mtb-induced DC migration and the ability of IL-12p40-deficient DCs to activate naive T cells. These data define a novel and fundamental role for IL-12p40 in the pathogen-induced activation of pulmonary DCs.

The role of cytokines in the initiation, expansion, and control of cellular immunity to tuberculosis.

Tuberculosis (TB) results from an interaction between a potent immune response and a chronically persistent pathogen. The ability of Mycobacterium tuberculosis (Mtb) to induce a strong immune response while being able to resist the ability of the host to clear bacteria provides an excellent tool with which to investigate the role of specific cytokine pathways on the induction, expansion, and control of the effector T-cell response. In this review, the role of interleukin-12p40 (IL-12p40), IL-12p70, IL-23, and IL-27 in the immune response to Mtb are described. We show that IL-12(p40)(2) acts to mediate the activation of dendritic cells to become responsive to homeostatic chemokines. We also show that IL-12p70 is required for the optimal interferon-gamma (IFN-gamma) T-cell response, which is required for control of Mtb growth. IL-23 can induce IFN-gamma responses in the lung if IL-12 is not present, but its major role is in supporting the IL-17 response within the lung. Neither IL-23 nor IL-17 is required for early control of Mtb in the lung. IL-23 and IL-17, however, can be instrumental in vaccine-induced protection. Finally, IL-27 limits protective immunity in the lung, but it is also required for long-term survival. These cytokines are therefore key players in the immune response to TB.

A Persistent Tuberculosis Outbreak in the UK Is Characterized by Hydrophobic fadB4-Deficient Mycobacterium tuberculosis That Replicates Rapidly in Macrophages.

The genetic diversity of Mycobacterium tuberculosis can influence disease severity and transmissibility. To better understand how this diversity influences individuals and communities, we phenotyped M. tuberculosis that was causing a persistent outbreak in the East Midlands, United Kingdom. Compared to nonoutbreak isolates, bacilli had higher lipid contents and more hydrophobic cell surfaces. In macrophage infection models, the bacteria increased more rapidly, provoked the enhanced accumulation of macrophage lipid droplets and enhanced the secretion of IL-1ß. Natural deletions in fadB4, nrdB, and plcC distinguished the outbreak isolates from other lineage 3 isolates in the region. fadB4 is annotated with a putative role in cell envelope biosynthesis, so the loss of this gene has the potential to alter the interactions of bacteria with immune cells. Reintroduction of fadB4 to the outbreak strain led to a phenotype that more closely resembled those of nonoutbreak strains. The improved understanding of the microbiological characteristics and the corresponding genetic polymorphisms that associate with outbreaks have the potential to inform tuberculosis control. IMPORTANCE Tuberculosis (TB) killed 1.5 million people in 2020 and affects every country. The extent to which the natural genetic diversity of Mycobacterium tuberculosis influences disease manifestation at both the individual and epidemiological levels remains poorly understood. Insights into how pathogen polymorphisms affect patterns of TB have the potential to translate into clinical and public health practice. Two distinct lineage 3 strains isolated from local TB outbreaks, one of which (CH) was rapidly terminated and the other of which (Lro) persistently transmitted for over a decade, provided us with an opportunity to study these issues. We compared genome sequences, microbiological characteristics, and early immune responses that were evoked upon infection. Our results indicate that the natural lack of fadB4 in the Lro strain contributes to its unique features.

Tc17, a unique subset of CD8 T cells that can protect against lethal influenza challenge.

We show here that IL-17-secreting CD4 T (Th)17 and CD8 T (Tc)17 effector cells are found in the lung following primary challenge with influenza A and that blocking Ab to IL-17 increases weight loss and reduces survival. Tc17 effectors can be generated in vitro using naive CD8 T cells from OT-I TCR-transgenic mice. T cell numbers expand 20-fold and a majority secretes IL-17, but little IFN-gamma. Many of the IL-17-secreting cells also secrete TNF and some secrete IL-2. Tc17 are negative for granzyme B, perforin message, and cytolytic activity, in contrast to Tc1 effectors. Tc17 populations express message for orphan nuclear receptor gammat and FoxP3, but are negative for T-bet and GATA-3 transcription factors. The FoxP3-positive, IL-17-secreting and IFN-gamma-secreting cells represent three separate populations. The IFN-gamma-, granzyme B-, FoxP3-positive cells and cells positive for IL-22 come mainly from memory cells and decrease in number when generated from CD44(low) rather than unselected CD8 T cells. Cells of this unique subset of CD8 effector T cells expand greatly after transfer to naive recipients following challenge and can protect them against lethal influenza infection. Tc17 protection is accompanied by greater neutrophil influx into the lung than in Tc1-injected mice, and the protection afforded by Tc17 effectors is less perforin but more IFN-gamma dependent, implying that different mechanisms are involved.