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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.

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-1β. Natural deletions in

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.

Little is known about T-cell responses during acute coronavirus disease-2019 (COVID-19). We measured T-cell interferon gamma (IFN-{gamma}) responses to spike 1 (S1), spike 2 (S2), nucleocapsid (N) and membrane (M) SARS-CoV-2 antigens using the T-SPOT(R) Discovery SARS-CoV-2 assay, a proven EliSPOT technology, in 114 hospitalised adult COVID-19 patients and assessed their association with clinical disease phenotype. T-SPOT(R) Discovery SARS-CoV-2 responses were detectable within 2 days of a positive PCR and did not correlate with vaccination status or symptom duration. Higher responses to S1 protein associated with a higher symptom burden, and serum IL-6 levels. Despite treatment with dexamethasone this subgroup was also at greater risk of requiring continuous positive airway pressure (CPAP) in the days following sampling. Higher T-cell responses measured using T-SPOT(R) Discovery SARS-CoV-2 associate with progressive disease in acute COVID-19 disease and may have utility as a prognostic biomarker that should be evaluated in larger cohorts.

Little is known about T-cell responses during acute coronavirus disease-2019 (COVID-19). We measured T-cell interferon gamma (IFN-γ) responses to spike 1 (S1), spike 2 (S2), nucleocapsid (N) and membrane (M) SARS-CoV-2 antigens using the T-SPOT® Discovery SARS-CoV-2 assay, a proven EliSPOT technology, in 114 hospitalised adult COVID-19 patients and assessed their association with clinical disease phenotype. T-SPOT® Discovery SARS-CoV-2 responses were detectable within 2 days of a positive PCR and did not correlate with vaccination status or symptom duration. Higher responses to S1 protein associated with a higher symptom burden, and serum IL-6 levels. Despite treatment with dexamethasone this subgroup was also at greater risk of requiring continuous positive airway pressure (CPAP) in the days following sampling. Higher T-cell responses measured using T-SPOT® Discovery SARS-CoV-2 associate with progressive disease in acute COVID-19 disease and may have utility as a prognostic biomarker that should be evaluated in larger cohorts.

Serodiagnosis of tuberculosis (TB) can be rapid, reliable and cost-effective if the issue of variable antibody responses of TB patients against different Mycobacterium tuberculosis (Mtb) antigens can be overcome by developing fusion proteins containing epitopes from multiple antigens of Mtb. In this study, Mtb antigens Rv1793, Rv2628, Rv2608 and a truncated variant produced by removing non-epitopic region from N-terminal of Rv2608 (tnRv2608), and the fusion protein Rv1793-Rv2628-tnRv2608 (TriFu64), were expressed in E. coli and purified. Plasma samples from TB patients characterized by sex, age and sputum/culture positivity, were used to compare the sensitivity of the single antigens with the fusion protein. Sensitivity of Rv1793, Rv2628 and Rv2608, was 27.8%, 39% and 36.3%, respectively. Truncation of Rv2608 increased sensitivity by approximately 35% in confirmed TB cases. Sensitivity of the fusion construct, TriFu64 increased to 66% with a specificity of 100%. Importantly, tnRv2608 was better able to detect sputum and culture negative patients, and this carried through to the fusion protein. We demonstrate that fusion of Mtb proteins ensures broad sensitivity across disease types, sex and age groups in a Pakistani population.

Background and Aims High intensity interval training (HIIT) has been shown to improve traditional and non-traditional cardiovascular risk factors in the general population and in chronic diseases. However, intense exercise has the potential to suppress immune function and promote systemic inflammation. Renal transplant recipients (RTRs) are immunologically vulnerable and therefore thorough understanding of the effects of different exercise regimes is required to inform exercise advice and guidelines. The aim of this study was to explore the immune and inflammatory effects of HIIT in comparison to moderate intensity continuous training (MICT) in RTRs. Method Renal transplant recipients (n = 24, age (years) = 48 ± 13, BMI = 27.2 ± 5.6 kg.m2, eGFR (mL·min· 1.73 m2) = 58 ± 19) were randomised into one of three thrice-weekly 8 week exercise programmes: HIIT A (16 min interval training with 4, 2 and 1 min intervals at 80%–90% of peak oxygen uptake (V̇O2 peak), HIIT B (4 × 4 min interval training at 80%–90% V̇O2peak) or MICT (∼40 min cycling at 50%–60% V̇O2peak). Peripheral blood mononuclear cells were isolated at baseline, mid-training, post-training and three months post-training and stained for lymphocyte and monocyte phenotypic and activation markers. Plasma cytokines (IL-6, IL-10 and TNF-α) were measured by affinity ELISA kits. Results Friedman’s ANOVA was used to assess within-group differences and a Kruskal-Wallis H test was used to assess between-group differences; any significant findings were followed up using a post-hoc test. There were no differences in circulating lymphocyte or monocyte lineage markers within or between groups over time (P > 0.05). Cytokines did not change in relation to exercise within or between groups over time (P > 0.05). Conclusion This is the first study to investigate the effects of HIIT on immune parameters and inflammation markers longitudinally in RTRs or other immunosuppressed populations. Our data do not indicate any evidence for adverse effects of HIIT on circulating lymphocytes, monocytes or cytokines and suggest that such exercise regimes may be immunologically safe for immunosuppressed patients. We recommend further investigations involving larger sample size to confirm our results.

Accurate prediction of which patient will progress from a sub-clinical Mycobacterium tuberculosis infection to active tuberculosis represents an elusive, yet critical, clinical research objective. From the individual perspective, progression can be considered to be the product of a series of unfortunate events or even a run of bad luck. Here, we identify the subtle physiological relationships that can influence the odds of progression to active TB and how this progression may reflect directed dysbiosis in a number of interrelated systems. Most infected individuals who progress to disease have apparently good immune responses, but these responses are, at times, compromised by either local or systemic environmental factors. Obvious disease promoting processes, such as tissue-damaging granulomata, usually manifest in the lung, but illness is systemic. This apparent dichotomy between local and systemic reflects a clear need to define the factors that promote progression to active disease within the context of the body as a physiological whole. We discuss aspects of the host environment that can impact expression of immunity, including the microbiome, glucocorticoid-mediated regulation, catecholamines and interaction between the gut, liver and lung. We suggest the importance of integrating precision medicine into our analyses of experimental outcomes such that apparently conflicting results are not contentious, but rather reflect the impact of these subtle relationships with our environment and microbiota.

Mycobacterium avium, one of the closest relatives of Mycobacterium tuberculosis (MTB), offers an advantage in studying MTB because of its tuberculosis-like effect in humans and host immune tolerance. This study examined the antimycobacterial action of ursolic acid and its regulation in macrophages during infection. Colony-forming units of the bacteria were determined in the cell lysate of macrophages and in the supernatant. The effect of ursolic acid on macrophages during infection was determined by analyzing the phosphorylation of the mitogen-activated protein kinase signaling pathway and the concentrations of tumor necrosis factor-α, interleukin-1β, interleukin-6, and nitrite. The colony-forming units analysis demonstrated that ursolic acid reduced the presence of Mycobacterium avium both intracellularly (in macrophages) and extracellularly. It decreased the levels of tumor necrosis factor-α and interleukin-6 but increased the concentrations of interleukin-1β and nitrite during infection. It also inhibited the phosphorylation of ERK1/2 but phosphorylated the C-Jun N-terminal kinase signaling pathway. The antimycobacterial effect of ursolic acid correlated with its ability to regulate the activation of macrophages. This dual ability made the ursolic acid-related elimination of the mycobacteria more effective.

Experimental mouse models of TB suggest that early events in the lung impact immunity. Early events in the human lung in response to TB are difficult to probe and their impact on disease outcome is unknown. We have shown in mouse that a secreted alternatively-spliced variant of IL-12Rβ1, lacking the transmembrane domain and termed ΔTM-IL-12Rβ1, promotes dendritic cell migration to the draining lymph node, augments T cell activation and limits dissemination ofM. tuberculosis(Mtb). We show here that CBA/J and C3H/HeJ mice (both highly susceptible to Mtb) express higher levels of ΔTM-IL-12Rβ1 than resistant C57BL6 mice and limit early dissemination of Mtb from the lungs. Both CD11c+ cells and T cells express ΔTM-IL-12Rβ1 in humans, and mice unable to make ΔTM-IL-12Rβ1 in either CD4 or CD11c expressing cells permit early dissemination from the lung. Analysis of publically available blood transcriptomes indicates that pulmonary TB is associated with high ΔTM-IL-12Rβ1 expression and that of all IL-12 related signals, the ΔTM-IL-12Rβ1 signal best predicts active disease. ΔTM-IL-12Rβ1 expression reflects the heterogeneity of latent TB infection and has the capacity to discriminate between latent and active disease. In a new Chinese TB patient cohort, ΔTM-IL-12Rβ1 effectively differentiates TB from latent TB, healthy controls and pneumonia patients. Finally, ΔTM-IL-12Rβ1 expression drops in drug-treated individuals in the UK and China where infection pressure is low. We propose that ΔTM-IL-12Rβ1 regulates early dissemination from the lung and that it has diagnostic potential and provides mechanistic insights into human TB.

A surprising immune twist for RORC The immune system needs its full array of soldiers—including cells and the molecules they secrete—to optimally protect the host. When this isn't the case, minor infections can become chronic or even deadly. Markle et al. report the discovery of seven individuals carrying loss-of-function mutations in RORC, which encodes the transcription factors RORγ and RORγT. These individuals lacked immune cells that produce the cytokine interleukin-17, causing them to suffer from chronic candidiasis. RORC-deficient individuals also exhibited impaired immunity to mycobacterium, probably due to reduced production of the cytokine interferon-γ, a molecule not known to require RORC for its induction. Science , this issue p. 606

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 (IL12Rβ1) that associates with IL12p40 and promotes the development of host-protective T H 1cells. Recently, we observed that il12rb1 —the mouse homolog of IL12RB1 —is alternatively spliced by leukocytes to produce a second isoform (IL12Rβ1ΔTM) that has biological properties distinct from IL12Rβ1. Although the expression of IL12Rβ1ΔTM is elicited by M. tuberculosis in vivo , and its overexpression enhances IL12p40 responsiveness in vitro , the contribution of IL12Rβ1ΔTM to controlling M. tuberculosis infection has not been tested. Here, we demonstrate that IL12Rβ1ΔTM 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 IL12Rβ1ΔTM-deficient animals are associated with decreased lymph node cellularity and a decline in T H 1 development. Collectively, these data support a model wherein IL12Rβ1ΔTM is a secreted product of il12rb1 that promotes resistance to M. tuberculosis infection by potentiating T H cells response to IL-12.

Mycobacterial tuberculosis remains a disease of major importance. In this Focus Review, Orme, Robinson and Cooper discuss lung immune responses to mycobacteria and describe how the bacterium can manipulate host immunity to its own ends. 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.

Chemokines and cytokines are critical for initiating and coordinating the organized and sequential recruitment and activation of cells into Mycobacterium tuberculosis -infected lungs. Correct mononuclear cellular recruitment and localization are essential to ensure control of bacterial growth without the development of diffuse and damaging granulocytic inflammation. An important block to our understanding of TB pathogenesis lies in dissecting the critical aspects of the cytokine/chemokine interplay in light of the conditional role these molecules play throughout infection and disease development. Much of the data highlighted in this review appears at first glance to be contradictory, but it is the balance between the cytokines and chemokines that is critical, and the “goldilocks” (not too much and not too little) phenomenon is paramount in any discussion of the role of these molecules in TB. Determination of how the key chemokines/cytokines and their receptors are balanced and how the loss of that balance can promote disease is vital to understanding TB pathogenesis and to identifying novel therapies for effective eradication of this disease.

IL-27 bridges innate and adaptive immunity by modulating cytokine production from myeloid cells and regulating Th cell differentiation. During bacterial infection, TLR4 triggering by LPS induces IL-27 production by monocytes and macrophages. We have previously shown that IL-27 can prime monocytes for LPS responsiveness by enhancing TLR4 expression and intracellular signaling. If unregulated, this could result in damaging inflammation, whereas on the other hand, this may also provide greater responses by inflammatory processes induced in response to bacterial pathogens. A key process in fine-tuning inflammatory responses is activation of the inflammasome, which ultimately results in IL-1β production. Herein, we investigated the molecular mechanisms by which IL-27 modulates LPS-induced IL-1β secretion in monocytes and macrophages. We found that when delivered simultaneously with LPS, IL-27 augments activation of caspase-1 and subsequent release of IL-1β. Furthermore, we determined that IL-27 primes cells for enhanced IL-1β production by up-regulating surface expression of TLR4 and P2X purinoceptor 7 (P2X7) for enhanced LPS and ATP signaling, respectively. These findings provide new evidence that IL-27 plays an important role in the proinflammatory capacity of monocytes and macrophages via enhancing IL-1β secretion levels triggered by dual LPS–ATP stimulation.

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.

Recent studies have revisited the roles of prime players in the immune response to tuberculosis (TB) and have highlighted novel functions of these players. Specifically, immunoregulatory mechanisms mediated by IFNγ have been delineated as well as a novel role for neutrophils in promoting antigen presentation. New insights into the interaction between the bacterium and phagocyte indicate that the bacterium actively promotes phagocyte necrosis rather than apoptosis and that this impacts generation of the acquired response. There are also many new examples of how the phagocyte responds to the bacteria and how it mediates control. The phenotype of protective T cells is also being re-examined. These developments provide promise for improved vaccine design and highlight the complexity of this disease.

Tuberculosis is the infectious disease of the human race, as it has been responsible for over one billion deaths in the last 200 years ([1][1]). Progress in controlling this disease is therefore a critical goal of the World Health Organization and other engaged, nongovernmental organizations ([2][2

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.

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.

Cells of the innate immune system produce cytokines and lipid mediators that strongly influence the outcome of mycobacterial infection. In the case of Mycobacterium tuberculosis, the lung is a critical site for this interaction. Here, we review current information on the role of the major innate cytokine pathways both in controlling initial infection as well as in promoting and maintaining adaptive T-cell responses that mediate host resistance or immunopathology. Understanding this important feature of the host-pathogen interaction can provide major insights into the mechanisms of virulence and can lead to new approaches for immunological intervention in tuberculosis and other mycobacterial diseases.

During inflammatory responses and wound healing, the conversion of soluble fibrinogen to fibrin, an insoluble extracellular matrix, long has been assumed to create a scaffold for the migration of leukocytes and fibroblasts. Previous studies concluded that fibrinogen is a necessary cofactor for mycobacterial trehalose 6,6′-dimycolate-induced responses, because trehalose dimycolate-coated beads, to which fibrinogen was adsorbed, were more inflammatory than those to which other plasma proteins were adsorbed. Herein, we investigate roles for fibrin(ogen) in anin vivomodel of mycobacterial granuloma formation and in infection withMycobacterium tuberculosis, the causative agent of tuberculosis. In wild-type mice, the subcutaneous injection of trehalose dimycolate-coated polystyrene microspheres, suspended within Matrigel, elicited a pyogranulomatous response during the course of 12 days. In fibrinogen-deficient mice, neutrophils were recruited but a more suppurative lesion developed, with the marked degradation and disintegration of the matrix. Compared to that in wild-type mice, the early formation of granulation tissue in fibrinogen-deficient mice was edematous, hypocellular, and disorganized. These deficiencies were complemented by the addition of exogenous fibrinogen. The absence of fibrinogen had no effect on cell recruitment or cytokine production in response to trehalose dimycolate, nor was there a difference in lung histopathology or overall bacterial burden in mice infected withMycobacterium tuberculosis. In this model, fibrin(ogen) was not required for cell recruitment, cytokine response, or response to infection, but it promoted granulation tissue formation and suppressed leukocyte necrosis.

Mycobacterium tuberculosis infection results in the generation of protective cellular immunity and formation of granulomatous structures in the lung. CXC chemokine ligand (CXCL)-13, CC chemokine ligand (CCL)-21 and CCL19 are constitutively expressed in the secondary lymphoid organs and play a dominant role in the homing of lymphocytes and dendritic cells. Although it is known that dendritic cell transport of M. tuberculosis from the lung to the draining lymph node is dependent on CCL19/CCL21, we show here that CCL19/CCL21 is also important for the accumulation of antigen-specific IFNγ-producing T cells in the lung, development of the granuloma, and control of mycobacteria. Importantly, we also show that CXCL13 is not required for generation of IFNγ responses, but is essential for the spatial arrangement of lymphocytes within granulomas, optimal activation of phagocytes and subsequent control of mycobacterial growth. Further, we show that these chemokines are also induced in the lung during the early immune responses following pulmonary M. tuberculosis infection. These results demonstrate that homeostatic chemokines perform distinct functions that cooperate to mediate effective expression of immunity against M. tuberculosis infection.

Tuberculosis is primarily a disease of the lung, and dissemination of the disease depends on productive infection of this critical organ. Upon aerosol infection with Mycobacterium tuberculosis (Mtb), the acquired cellular immune response is slow to be induced and to be expressed within the lung. This slowness allows infection to become well established; thus, the acquired response is expressed in an inflammatory site that has been initiated and modulated by the bacterium. Mtb has a variety of surface molecules that interact with the innate response, and this interaction along with the autoregulation of the immune response by several mechanisms results in less-than-optimal control of bacterial growth. To improve current vaccine strategies, we must understand the factors that mediate induction, expression, and regulation of the immune response in the lung. We must also determine how to induce both known and novel immunoprotective responses without inducing immunopathologic consequences.

IL-17 can impact health in a variety of ways. It is protective for some pathogens but it is also associated with tissue damaging inflammation. By examining the role of IL-17 in a variety of bacterial infections the mechanisms by which this cytokine mediates both protection and damage can be dissected. A key element in understanding the role of this cytokine is determining where and when it is acting. Dissecting its essential protective role from its immunopathologic role will allow for improved intervention in both acute and chronic disease.

At the temperature of its flea vector (approximately 20-30 degrees C), the causative agent of plague, Yersinia pestis, expresses a profile of genes distinct from those expressed in a mammalian host (37 degrees C). When dendritic cells (DC) are exposed to Y. pestis grown at 26 degrees C (Y. pestis-26 degrees), they secrete copious amounts of IL-12p40 homodimer (IL-12(p40)(2)). In contrast, when DCs are exposed to Y. pestis grown at 37 degrees C (Y. pestis-37 degrees), they transcribe very little IL-12p40, which is secreted as IL-12p40 monomer (IL-12p40). Y. pestis-26 degrees also induces migration of DCs to the homeostatic chemokine CCL19, whereas Y. pestis-37 degrees does not; migratory DCs are positive for IL-12p40 transcription and secrete mostly IL-12(p40)(2); DCs lacking IL-12p40 do not migrate. Expression of acyltransferase LpxL from Escherichia coli in Y. pestis-37 degrees results in the production of a hexa-acylated lipid A, also seen in Y. pestis-26 degrees, rather than tetra-acylated lipid A normally seen in Y. pestis-37 degrees. The LpxL-expressing Y. pestis-37 degrees promotes DC IL-12(p40)(2) production and induction of DC migration. In addition, absence of TLR4 ablates production of IL-12(p40)(2) in DC exposed to Y. pestis-26 degrees. The data demonstrate the molecular pathway by which Y. pestis evades induction of early DC activation as measured by migration and IL-12(p40)(2) production.

Tuberculosis (TB) has plagued mankind for millennia yet is classified as an emerging infectious disease, because its prevalence in the human population continues to increase. Immunity to TB depends critically on the generation of effective CD4(+) T-cell responses. Sterile immunity has not been achieved through vaccination, although early T-cell responses are effective in controlling steady-state infection in the lungs. Although such early T-cell responses are clearly protective, the initiation of the Mycobacterium tuberculosis (Mtb) T-cell response occurs much later than is the case following other aerogenic infections. This fact suggests that there is a critical period, before the activation of the T-cell response, in which Mtb is able to establish infection. An understanding of the factors that regulate early T-cell activation should, therefore, lead to better control of the disease. This review discusses recent work that has investigated the early development of T-cell immunity following Mtb infection in the mouse.

CD8+ T cells are a major source of IFN-γ, a key effector cytokine in immune responses against many viruses and protozoa. Although the transcription factor T-bet is required for IFN-γ expression in CD4+ T cells, it is reportedly dispensable in CD8+ T cells, where the transcription factor Eomesodermin is thought to be sufficient. The diverse functions of IFN-γ are mediated through the IFN-γR and STAT1. In CD4+ T cells, STAT1 appears to be critical for the activation of T-bet and IFN-γ, suggesting an IFN-γ-dependent positive feedback loop. However, STAT1 can also be activated by other cytokines, including IL-27. In the present study we show that, in contrast to in vitro conditions and the prevailing paradigm, T-bet is critical for the in vivo IFN-γ production by CD8+ T cells upon infection of mice with diverse pathogens. Whereas IFN-γR signals are dispensable for the T-bet-dependent IFN-γ production, direct IL-27Rα signals are critical.

IL-12p40 is known as a component of the bioactive cytokines interleukin (IL)-12 and IL-23 but it is not widely recognized as having intrinsic functional activity. Recent publications have altered this perception and support an independent role for IL-12p40. IL-12p40 is induced in excess over the other subunits of IL-12 and IL-23 and can exist in a monomeric or homodimeric form. Its most widely appreciated function is to provide a negative feedback loop by competitively binding to the IL-12 receptor. However, IL-12p40 acts as a chemoattractant for macrophages and promotes the migration of bacterially stimulated dendritic cells. It is associated with several pathogenic inflammatory responses such as silicosis, graft rejection and asthma but it is also protective in a mycobacterial model. An appreciation of the independent function of IL-12p40 is important for improving our understanding of both protective and pathogenic immune responses.

To elucidate the little-known bioenergetic pathways of host immune cells in tuberculosis, a granulomatous disease caused by the intracellular pathogen Mycobacterium tuberculosis, we characterized infected murine lung tissue by transcriptomic profiling and confocal imaging. Transcriptomic analysis revealed changes of host energy metabolism during the course of infection that are characterized by upregulation of key glycolytic enzymes and transporters for glucose uptake and downregulation of enzymes participating in the tricarboxylic acid cycle and oxidative phosphorylation. Consistent with elevated glycolysis, we also observed upregulation of a transporter for lactate secretion and a V type H+ -ATPase involved in cytosolic pH homeostasis. Transcription profiling results were corroborated by immunofluorescence microscopy showing increased expression of key glycolytic enzymes in macrophages and T cells in granulomatous lesions. Moreover, we found increased mRNA and protein levels in macrophages and T cells of hypoxia inducible factor 1 alpha (HIF-1α), the regulatory subunit of HIF-1, a master transcriptional regulator. Thus, our findings suggest that immune cells predominantly utilize aerobic glycolysis in response to M. tuberculosis infection. This bioenergetic shift is similar to the Warburg effect, the metabolic signature of cancer cells. Finding immunometabolic changes during M. tuberculosis infection opens the way to new strategies for immunotherapy against tuberculosis.

The fate of infected macrophages is a critical aspect of immunity to mycobacteria. By depriving the pathogen of its intracellular niche, apoptotic death of the infected macrophage has been shown to be an important mechanism to control bacterial growth. Here, we show that IL-17 inhibits apoptosis of Mycobacterium bovis BCG- or Mycobacterium tuberculosis-infected macrophages thus hampering their ability to control bacterial growth. Mechanistically, we show that IL-17 inhibits p53, and impacts on the intrinsic apoptotic pathway, by increasing the Bcl2 and decreasing Bax expression, decreasing cytochrome c release from the mitochondria, and inhibiting caspase-3 activation. The same effect of IL-17 was observed in infected macrophages upon blockade of p53 nuclear translocation. These results reveal a previously unappreciated role for the IL-17/p53 axis in the regulation of mycobacteria-induced apoptosis and can have important implications in a broad spectrum of diseases where apoptosis of the infected cell is an important host defense mechanism., Fundação para a Ciência e Tecnologia, Portugal. Project grants: PTDC/SAU-MII/101977/2008 (to AGC), PTDC/BIA-BCM/102776/2008 (to MS) and HMSP-ICT/0024/2010 (to RA) and co-funded by Programa Operacional Regional do Norte (ON.2 – O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER). Personal Grants: SFRH/BPD/33036/2006 to AC; SFRH/BD/33573/2009

Summary 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.

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.

Testing of drugs or vaccines in animal models prior to studies in the human population are essential to avoid safety problems in the field. In this chapter, the mouse, guinea pig, rabbit, and nonhuman primate models are discussed. Each model has its strengths and weaknesses, and the choice of the most appropriate model for a study depends on a variety of factors, including cost, available housing, and the question being addressed. The mouse model provides an economical and easily manipulated tool with which to determine the role of specific host or bacterial components in the pathogenesis of tuberculosis. The development of a chronic infection is common to most mammals infected with Mycobacterium tuberculosis. One important factor in using the aerosol model to assess the pathogenesis of M. tuberculosis is the use of bacterial cultures with high viability. Caseous necrosis in the form of small, spherical tubercles is a hallmark of human tuberculosis; indeed, it is the pathologic entity from which the disease derives its name. Nonhuman primates have been used in tuberculosis research for many decades, although cost and containment requirements have reduced the use of this model substantially in the last 30 years. The increasing sophistication of the animal models will lead the way to new findings of great importance in tuberculosis.

Fibrinogen-like protein 2 (Fgl2, fibroleukin) is a leukocyte product that exhibits significant homology to secreted proteins of diverse function, including growth factors, lectins, and components of extracellular matrix. Prior studies found that Fgl2 is IFNγ-inducible, possesses direct coagulant activity, and inhibits T cell proliferation and dendritic cell maturationin vitro. Here, we demonstrate that Fgl2 expression is up-regulated during type 1 immunityin vivoand establish that such up-regulation is IFNγ-, signal transducer and activation of transcription protein 1-, and IFN response factor 1-dependent. To investigate functional roles for Fgl2 during type 1 immunity, we generated Fgl2-deficient mice. Those animals are born at predicted Mendelian frequencies, appear overtly healthy, and contain normal numbers and frequencies of lymphoid cells. Although Fgl2 is IFNγ-inducible and putatively regulates T cell activation/proliferation, we demonstrate that Fgl2-deficient and control mice exhibit similar degrees of T cell expansion, immunopathology, and/or pathogen burdens during protozoan (Toxoplasma gondii), bacterial (Yersinia enterocolitica, Listeria monocytogenes, andMycobacterium tuberculosis), and viral (murine γ-herpesvirus-68 and Sendai) infections. Fgl2-deficient mice also reject allografts with similar kinetics as control mice. Moreover, despite prior reports that Fgl2 functions as a procoagulant enzyme, we demonstrate that Fgl2-deficient and control mice produce similar levels of fibrin, a product of the coagulation cascade, duringT. gondiiinfection and allograft rejection. Together, our findings suggest that Fgl2, although highly conserved and IFNγ-inducible, is not a critical mediator of either type 1 immunity or immune-associated coagulant activity.

Setting : Members of the Mycobacterium avium complex (MAC) are responsible for mycobacterial disease in children, the aged and in immunocompromised individuals. The complex consists of different species, serovars and morphologic forms that vary in virulence. One isolate of the MAC is currently being sequenced (MAC 104) and was chosen based on its derivation from an AIDS patient and the fact that it could be genetically manipulated. Objective : MAC 104 was therefore analyzed for virulence, colony morphotype and expression of the glycopeptidolipid (GPL) responsible for serotying differences and the rough to smooth morphological switch. Results : The isolate was found to be virulent in the murine model of low-dose aerosol infection in that it could colonize the lung, proliferate within the tissue and disseminate to other organs. MAC 104 expressed a variety of colony morphotypes, the most prevalent of which were smooth opaque, smooth transparent and rough. All three morphotypes could persist in the lung; however, the transparent and rough morphotypes grew more rapidly in vivo . The rough morphotype was unusual in that it expressed an atypical form of the GPL usually absent from rough morphotypes. Conclusion : This characterization complements the genome data and confirms that MAC 104 behaves similarly to other MAC isolates.

Mycobacterium leprae infection was evaluated in interferon-gamma knockout (GKO) mice. At 4 months, growth of the bacilli in the footpads of GKO mice plateaued a log(10) higher than that in control mice. Control mice exhibited mild lymphocytic and histiocytic infiltrates, whereas GKO mice developed large, unorganized infiltrates of epithelioid macrophages and scattered CD4 and CD8 T cells. Flow cytometric analysis of popliteal lymph node cells demonstrated similar profiles of T cells; however, GKO cells exhibited an elevated proliferative response to M. leprae antigen. Expression of inducible nitric oxide synthase mRNA was decreased in GKO mice, whereas macrophage inflammatory protein-1alpha and interleukin-4 and -10 mRNA expression were augmented. Control and GKO activated macrophages inhibited bacterial metabolism and produced nitrite. Thus, although deficient in an important Th1 cytokine, GKO mice possess compensatory mechanisms to control M. leprae growth and feature elements resembling mid-borderline leprosy in humans.

Recent evidence suggests that absence of the IL-12p40 subunit is more detrimental to the generation of protective responses than is the absence of the p35 subunit. To determine whether this is the case in tuberculosis, both p35 and p40 knockout mice were infected with Mycobacterium tuberculosis. Mice lacking the p40 subunit were highly susceptible to increased bacterial growth, exhibited reduced production of IFN-γ, and had increased mortality. In contrast, mice lacking the p35 subunit exhibited a moderate ability to control bacterial growth, were able to generate Ag-specific IFN-γ responses, and survived infection longer. The superior Ag-specific responses of the p35 gene-disrupted mice, when compared with the p40 gene-disrupted mice, suggest that the p40 subunit may act other than as a component of IL-12. A candidate molecule capable of driving the protective responses in the p35 gene-disrupted mice is the novel cytokine IL-23. This cytokine is composed of the IL-12 p40 subunit and a p19 subunit. In support of a role for this cytokine in protective responses to M. tuberculosis, we determined that the p19 subunit is induced in the lungs of infected mice.

Of the two common morphotypes of Mycobacterium avium, designated smooth transparent (SmT) or smooth opaque (SmO), the SmO morphotype is avirulent, whereas the SmT morphotype is virulent. The role of the host macrophage in determining these different virulence phenotypes was analyzed using an in vitro model of macrophage infection. Initial studies confirmed previous reports of the increased ability of the SmT bacteria to grow in macrophages; this increased virulence correlated with reduced induction of inflammatory cytokines. Examination of the response of the mitogen-activated protein kinase (MAPK) pathway following infection with either morphotype revealed that all three members of the MAPK pathway were activated. Pharmacologic inhibition of either the extracellular signal-regulated kinase (ERK) or p38MAPK pathways resulted in distinct consequences for the growth of the two morphotypes. In particular, inhibition of the p38MAPK resulted in attenuated growth of the SmT morphotype, which correlated with reduced PGE2 production. Inhibition of cyclooxygenase 2 by indomethacin also inhibited growth of SmT, substantiating the role for PGE2 in promoting the growth of SmT. In contrast, SmO induction of the ERK pathway was increased compared with the SmT morphotype, and inhibition of ERK resulted in decreased TNF-α synthesis and enhanced SmO growth. Pharmacologic inhibitors of the MAPK pathway were present for only the first 4 h of infection and yet had consequences for bacterial growth at 7 days. Therefore, the data suggest that induction of the MAPK pathway during uptake of bacteria is instrumental in determining the eventual fate of the bacteria.

Human inborn errors of immunity mediated by the cytokines interleukin-17A and interleukin-17F (IL-17A/F) underlie mucocutaneous candidiasis, whereas inborn errors of interferon-γ (IFN-γ) immunity underlie mycobacterial disease. We report the discovery of bi-allelic RORC loss-of-function mutations in seven individuals from three kindreds of different ethnic origins with both candidiasis and mycobacteriosis. The lack of functional RORγ and RORγT isoforms resulted in the absence of IL-17A/F-producing T cells in these individuals, probably accounting for their chronic candidiasis. Unexpectedly, leukocytes from RORγ- and RORγT-deficient individuals also displayed an impaired IFN-γ response to Mycobacterium. This principally reflected profoundly defective IFN-γ production by circulating γδ T cells and CD4(+)CCR6(+)CXCR3(+) αβ T cells. In humans, both mucocutaneous immunity to Candida and systemic immunity to Mycobacterium require RORγ, RORγT, or both.

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.

The cytokine interleukin-12 (IL-12) was thought to have a central role in T cell-mediated responses in inflammation for more than a decade after it was first identified. Discovery of the cytokine IL-23, which shares a common p40 subunit with IL-12, prompted efforts to clarify the relative contribution of these two cytokines in immune regulation. Ustekinumab, a therapeutic agent targeting both cytokines, was recently approved to treat psoriasis and psoriatic arthritis, and related agents are in clinical testing for a variety of inflammatory disorders. Here we discuss the therapeutic rationale for targeting these cytokines, the unintended consequences for host defense and tumor surveillance and potential ways in which these therapies can be applied to treat additional immune disorders.

It is unclear how CD4 T cell memory formation is regulated following pathogen challenge, and when critical mechanisms act to determine effector T cell fate. Here, we report that following influenza infection most effectors require signals from major histocompatibility complex class II molecules and CD70 during a late window well after initial priming to become memory. During this timeframe, effector cells must produce IL-2 or be exposed to high levels of paracrine or exogenously added IL-2 to survive an otherwise rapid default contraction phase. Late IL-2 promotes survival through acute down regulation of apoptotic pathways in effector T cells and by permanently upregulating their IL-7 receptor expression, enabling IL-7 to sustain them as memory T cells. This new paradigm defines a late checkpoint during the effector phase at which cognate interactions direct CD4 T cell memory generation.

Loss of IL-27R on T cells results in increased protection from Mycobacterium tuberculosis. Torrado et al. demonstrate that IL-27R−/− T cells show improved fitness that is associated with decreased expression of cell death molecules, maintenance of IL-2 production, and preferential accumulation in the lung parenchyma and around infected macrophages., CD4+ T cells mediate protection against Mycobacterium tuberculosis (Mtb); however, the phenotype of protective T cells is undefined, thereby confounding vaccination efforts. IL-27 is highly expressed during human tuberculosis (TB), and absence of IL-27R (Il27ra) specifically on T cells results in increased protection. IL-27R deficiency during chronic Mtb infection does not impact antigen-specific CD4+ T cell number but maintains programmed death-1 (PD-1), CD69, and CD127 expression while reducing T-bet and killer cell lectin-like receptor G1 (KLRG1) expression. Furthermore, T-bet haploinsufficiency results in failure to generate KLRG1+, antigen-specific CD4+ T cells, and in improved protection. T cells in Il27ra−/− mice accumulate preferentially in the lung parenchyma within close proximity to Mtb, and antigen-specific CD4+ T cells lacking IL-27R are intrinsically more fit than intact T cells and maintain IL-2 production. Improved fitness of IL-27R–deficient T cells is not associated with increased proliferation but with decreased expression of cell death–associated markers. Therefore, during Mtb infection, IL-27R acts intrinsically on T cells to limit protection and reduce fitness, whereas the IL-27R–deficient environment alters the phenotype and location of T cells. The significant expression of IL-27 in TB and the negative influence of IL-27R on T cell function demonstrate the pathway by which this cytokine/receptor pair is detrimental in TB.

Mycobacterium bovis Bacille Calmette-Guerin (BCG) is the only vaccine in use to prevent Mycobacterium tuberculosis (Mtb) infection. Here we analyzed the protective efficacy of BCG against Mtb challenges 21 or 120 days after vaccination. Only after 120 days post-vaccination were mice able to efficiently induce early Mtb growth arrest and maintain long-lasting control of Mtb. This protection correlated with the accumulation of CD4(+) T cells expressing IL-17(+)TNF(+)IL-2(+). In contrast, mice challenged with Mtb 21 days after BCG vaccination exhibited only a mild and transient protection, associated with the accumulation of CD4(+) T cells that were mostly IFN-gamma(+)TNF(+) and to a lesser extent IFN-gamma(+)TNF(+)IL-2(+). These data suggest that the memory response generated by BCG vaccination is functionally distinct depending upon the temporal proximity to BCG vaccination. Understanding how these responses are generated and maintained is critical for the development of novel vaccination strategies against tuberculosis. Copyright 2014 Elsevier Ltd. All rights reserved., We thank the ICVS animal facility personnel for excellent animal husbandry. This work was supported by Fundacao para a Ciencia e Tecnologia, Portugal and cofunded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), Quadro de Referencia Estrategico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER) project grants PTDC/SAU-MII/101977/2008, PTDC/BIA-BCM/102776/2008, and from Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013). AMC was funded by the National Institute of Allergy and Infectious Diseases at the National Institutes of Health grant AI46530. A.C. received a personal FCT grant SFRH/BPD/3306/2007 and M.S. is an FCT Investigator fellow.