HIV infection impairs the host response to Mycobacterium tuberculosis infection by altering surfactant protein D function in the human lung alveolar mucosa.

Tuberculosis is the leading cause of death for people living with HIV (PLWH). We hypothesized that altered functions of innate immune components in the human alveolar lining fluid of PLWH (HIV-ALF) drive susceptibility to Mycobacterium tuberculosis (M.tb) infection. Our results indicate a significant increase in oxidation of innate proteins and chemokine levels and significantly lower levels and function of complement components and Th1/Th2/Th17 cytokines in HIV-ALF versus control-ALF (non-HIV-infected people). We further found a deficiency of surfactant protein D (SP-D) and reduced binding of SP-D to M.tb that had been exposed to HIV-ALF. Primary human macrophages infected with M.tb exposed to HIV-ALF were significantly less capable of controlling the infection, which was reversed by SP-D replenishment in HIV-ALF. Thus, based on the limited number of participants in this study, our data suggest that PLWH without antiretroviral therapy (ART) have declining host innate defense function in their lung mucosa, thereby favoring M.tb and potentially other pulmonary infections.

Human alveolar lining fluid from the elderly promotes Mycobacterium tuberculosis intracellular growth and translocation into the cytosol of alveolar epithelial cells.

The elderly population is highly susceptible to developing respiratory diseases, including tuberculosis, a devastating disease caused by the airborne pathogen Mycobacterium tuberculosis (M.tb) that kills one person every 18 seconds. Once M.tb reaches the alveolar space, it contacts alveolar lining fluid (ALF), which dictates host-cell interactions. We previously determined that age-associated dysfunction of soluble innate components in human ALF leads to accelerated M.tb growth within human alveolar macrophages. Here we determined the impact of human ALF on M.tb infection of alveolar epithelial type cells (ATs), another critical lung cellular determinant of infection. We observed that elderly ALF (E-ALF)-exposed M.tb had significantly increased intracellular growth with rapid replication in ATs compared to adult ALF (A-ALF)-exposed bacteria, as well as a dampened inflammatory response. A potential mechanism underlying this accelerated growth in ATs was our observation of increased bacterial translocation into the cytosol, a compartment that favors bacterial replication. These findings in the context of our previous studies highlight how the oxidative and dysfunctional status of the elderly lung mucosa determines susceptibility to M.tb infection, including dampening immune responses and favoring bacterial replication within alveolar resident cell populations, including ATs, the most abundant resident cell type within the alveoli.

Acute inflammation alters lung lymphocytes and potentiates innate-like behavior in young mouse lung CD8 T cells, resembling lung CD8 T cells from old mice.

Inflammation plays a significant role in lung infection including that caused by Mycobacterium tuberculosis, in which both adaptive and innate lymphocytes can affect infection control. How inflammation affects infection is understood in a broad sense, including inflammaging (chronic inflammation) seen in the elderly, but the explicit role that inflammation can play in regulation of lymphocyte function is not known. To fill this knowledge gap, we used an acute lipopolysaccharide (LPS) treatment in young mice and studied lymphocyte responses, focusing on CD8 T cell subsets. LPS treatment decreased the total numbers of T cells in the lungs of LPS mice while also increasing the number of activated T cells. We demonstrate that lung CD8 T cells from LPS mice became capable of an antigen independent innate-like IFN-γ secretion, dependent on IL-12p70 stimulation, paralleling innate-like IFN-γ secretion of lung CD8 T cells from old mice. Overall, this study provides information on how acute inflammation can affect lymphocytes, particularly CD8 T cells, which could potentially affect immune control of various disease states.

Differential Role of Type 2 Diabetes as a Risk Factor for Tuberculosis in the Elderly versus Younger Adults.

The elderly are understudied despite their high risk of tuberculosis (TB). We sought to identify factors underlying the lack of an association between TB and type 2 diabetes (T2D) in the elderly, but not adults. We conducted a case-control study in elderly (≥65 years old; ELD) vs. younger adults (young/middle-aged adults (18-44/45-64 years old; YA|MAA) stratified by TB and T2D, using a research study population (n = 1160) and TB surveillance data (n = 8783). In the research study population the adjusted odds ratio (AOR) of TB in T2D was highest in young adults (AOR 6.48) but waned with age becoming non-significant in the elderly. Findings were validated using TB surveillance data. T2D in the elderly (vs. T2D in younger individuals) was characterized by better glucose control (e.g., lower hyperglycemia or HbA1c), lower insulin resistance, more sulphonylureas use, and features of less inflammation (e.g., lower obesity, neutrophils, platelets, anti-inflammatory use). We posit that differences underlying glucose dysregulation and inflammation in elderly vs. younger adults with T2D, contribute to their differential association with TB. Studies in the elderly provide valuable insights into TB-T2D pathogenesis, e.g., here we identified insulin resistance as a novel candidate mechanism by which T2D may increase active TB risk.

Recall responses in the lung environment are impacted by age in a pilot study of Mycobacterium bovis-BCG vaccinated rhesus macaques.

Age-related changes in the immune system increase susceptibility to infectious diseases. Vaccines are an important tool to prevent infection or boost immunological memory; however, vaccines are less effective in aged individuals. In order to protect our aging population from the threat of infectious diseases, we must gain a better understanding of age-related alterations in the immune response at the site of infection. The lung is one site of frequent infection in older individuals. In this study, we expanded on our previous work to study vaccine-induced immune responses in the local lung environment in a pilot study of aged rhesus macaques. To do this, we developed an in vivo model to probe recall responses to tuberculin challenge in the lungs 8 weeks and 16 weeks post-Mycobacterium bovis BCG vaccination by performing targeted bronchoalveolar lavages. In parallel, we determined peripheral blood responses in vaccinated animals to compare systemic and local tissue responses to tuberculin challenge. We found that following lung tuberculin challenge 8 weeks post-vaccination, aged animals had reduced T cell responses, particularly within the CD8+ T cell compartment. Aged animals had decreased CD8+ effector and memory T cell recall responses and less activated CD8+ T cells. This diminished lung CD8+ T cell response in aged animals was maintained over time. Despite changes in the CD8+ T cell compartment, lung CD4+ T cell responses were similar between age groups. In the peripheral blood, we observed age-related changes in immune cell populations and plasma levels of immune mediators that were present prior to vaccination. Lastly, we found that peripheral blood mononuclear cells from aged BCG-vaccinated animals were functional in their response to antigen stimulation, behaving in a similar manner to those from their adult counterparts. These systemic observations were similar to those found in our previous study of BCG-vaccinated baboons, supporting the notion that tissue immune responses, and not systemic responses, to vaccination and challenge are impaired with age. These findings expand on our previous work to show that in addition to the skin, age-related changes in the lung environment impact recall immune responses to vaccination and challenge. The impact of age on local tissue responses to infectious challenge should be accounted for in the development of therapeutics or medical interventions aimed at boosting immune recall responses of aged individuals.

Interferon gamma release assays for detection of latent Mycobacterium tuberculosis in older Hispanic people.

BACKGROUND: Interferon gamma release assays (IGRAs) are used to detect latent Mycobacterium tuberculosis (M.tb) infection (LTBI) in adults, but their performance in older people is not well-established. We evaluated IGRAs for LTBI detection in older Hispanic recent TB contacts (ReC) or community controls (CoC). METHODS: Cross-sectional assessment of LTBI with T-SPOT.TB and/or QuantiFERON-Gold in-tube or -Plus assay in older (≥60 years) and adult (18-50 years) Hispanic people. RESULTS: We enrolled 193 CoC (119 adults, 74 older persons) and 459 ReC (361 adults, 98 older persons). LTBI positivity increased with age in CoC (19%-59%, P<0.001), but was similar in ReC (59%-69%, P=0.329). Older people had lower concordance between IGRAs (kappa 0.465 vs 0.688 in adults) and more inconclusive results (indeterminate/borderline 11.6% vs 5.8% in adults, P=0.012). With simultaneous IGRAs, inconclusive results were resolved as positive or negative with the other IGRA. The magnitude of response to M.tb peptides in IGRAs was similar among age groups, but responsiveness to mitogens was lower in older people. CONCLUSIONS: IGRAs are suitable for LTBI detection in older people. Discordant and inconclusive findings are more prevalent in older people, but results are resolved when IGRA is repeated with a different IGRA test.

Acute Inflammation Confers Enhanced Protection against Mycobacterium tuberculosis Infection in Mice.

Inflammation plays a crucial role in the control of Mycobacterium tuberculosis infection. In this study, we demonstrate that an inflammatory pulmonary environment at the time of infection mediated by lipopolysaccharide treatment in mice confers enhanced protection against M. tuberculosis for up to 6 months postinfection. This early and transient inflammatory environment was associated with a neutrophil and CD11b+ cell influx and increased inflammatory cytokines. In vitro infection demonstrated that neutrophils from lipopolysaccharide-treated mice exhibited increased association with M. tuberculosis and had a greater innate capacity for killing M. tuberculosis. Finally, partial depletion of neutrophils in lipopolysaccharide-treated mice showed an increase in M. tuberculosis burden, suggesting neutrophils played a part in the protection observed in lipopolysaccharide-treated mice. These results indicate a positive role for an inflammatory environment in the initial stages of M. tuberculosis infection and suggest that acute inflammation at the time of M. tuberculosis infection can positively alter disease outcome. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis disease, is estimated to infect one-fourth of the world's population and is one of the leading causes of death due to an infectious disease worldwide. The high-level variability in tuberculosis disease responses in the human populace may be linked to immune processes related to inflammation. In many cases, inflammation appears to exasperate tuberculosis responses; however, some evidence suggests inflammatory processes improve control of M. tuberculosis infection. Here, we show an acute inflammatory stimulus in mice provides protection against M. tuberculosis for up to 6 months, suggesting acute inflammation can positively affect M. tuberculosis infection outcome.

A prospective cross-sectional study of tuberculosis in elderly Hispanics reveals that BCG vaccination at birth is protective whereas diabetes is not a risk factor.

BACKGROUND: Aging increases the risk of tuberculosis (TB) and its adverse outcomes, but most studies are based on secondary analyses, and few are in Hispanics. Diabetes is a risk factor for TB in adults, but its contribution in the elderly is unknown. We aimed to identify the role of diabetes and other risk factors for TB in elderly Hispanics. METHODS: Cross-sectional study among newly-diagnosed TB patients, recent contacts (ReC), or community controls (CoC) totaling 646 participants, including 183 elderly (>60 years; 43 TB, 80 ReC, 60 CoC) and 463 adults (18 to 50 years; 80 TB, 301 ReC and 82 CoC). Host characteristics associated with TB and latent Mycobacterium tuberculosis infection (LTBI) were identified in the elderly by univariable and confirmed by multivariable logistic regression. RESULTS: LTBI was more prevalent among the elderly CoC (55% vs. 23.2% in adults; p<0.001), but not in ReC (elderly 71.3% vs. adult 63.8%); p = 0.213). Risk factors for TB in the elderly included male sex (adj-OR 4.33, 95% CI 1.76, 10.65), smoking (adj-OR 2.55, 95% CI 1.01, 6.45) and low BMI (adj-OR 12.34, 95% CI 4.44, 34.33). Unexpectedly, type 2 diabetes was not associated with TB despite its high prevalence (adj-OR 0.38, 95% CI 0.06, 2.38), and BCG vaccination at birth was protective (adj-OR 0.16, 95% CI 0.06, 0.45). CONCLUSIONS: We report novel distinctions in TB risk factors in the elderly vs. adults, notably in diabetes and BCG vaccination at birth. Further studies are warranted to address disparities in this vulnerable, understudied population.

Local immune responses to tuberculin skin challenge in Mycobacterium bovis BCG-vaccinated baboons: a pilot study of younger and older animals.

Individuals over the age of 65 are highly susceptible to infectious diseases, which account for one-third of deaths in this age group. Vaccines are a primary tool to combat infection, yet they are less effective in the elderly population. While many groups have aimed to address this problem by studying vaccine-induced peripheral blood responses in the elderly, work from our lab and others demonstrate that immune responses to vaccination and infectious challenge may differ between tissue sites and the periphery. In this pilot study, we established an in vivo delayed-type hypersensitivity model of Mycobacterium bovis BCG vaccination and tuberculin skin test in two adult and two aged baboons. Vaccination generates BCG-specific immune cells that are recruited to the skin upon tuberculin challenge. We tested short term recall responses (8 weeks post-vaccination) and long term recall responses (25 weeks post-vaccination) by performing skin punch biopsies around the site of tuberculin injection. In short term recall responses, we found increased oxidation and decreased production of immune proteins in aged baboon skin at the site of TST challenge, in comparison to adult skin. Differences between adult and aged animals normalized in the long term response to tuberculin. In vitro, aged peripheral blood mononuclear cells had increased migration and functional responses to antigen-specific stimulation, suggesting that age-related changes in the tissue in vivo impairs aged immune recall responses to antigenic challenge. These findings highlight the impact of age-associated changes in the local tissue environment in memory recall responses, which may be more broadly applied to the study of other tissues. Moreover, these findings should be considered in future studies aimed at understanding and improving aging immune responses to vaccination and tissue challenge.

Formation of Lung Inducible Bronchus Associated Lymphoid Tissue Is Regulated by Mycobacterium tuberculosis Expressed Determinants.

Mycobacterium tuberculosis (Mtb) is the causative agent of the infectious disease tuberculosis (TB), which is a leading cause of death worldwide. Approximately one fourth of the world's population is infected with Mtb. A major unresolved question is delineating the inducers of protective long-lasting immune response without inducing overt, lung inflammation. Previous studies have shown that the presence of inducible Bronchus-Associated Lymphoid Tissue (iBALT) correlate with protection from Mtb infection. In this study, we hypothesized that specific Mtb factors could influence the formation of iBALT, thus skewing the outcome of TB disease. We infected non-human primates (NHPs) with a transposon mutant library of Mtb, and identified specific Mtb mutants that were over-represented within iBALT-containing granulomas. A major pathway reflected in these mutants was Mtb cell wall lipid transport and metabolism. We mechanistically addressed the function of one such Mtb mutant lacking mycobacteria membrane protein large 7 (MmpL7), which transports phthiocerol dimycocerosate (PDIM) to the mycobacterial outer membrane (MOM). Accordingly, murine aerosol infection with the Mtb mutant Δmmpl7 correlated with increased iBALT-containing granulomas. Our studies showed that the Δmmpl7 mutant lacking PDIMs on the surface overexpressed diacyl trehaloses (DATs) in the cell wall, which altered the cytokine/chemokine production of epithelial and myeloid cells, thus leading to a dampened inflammatory response. Thus, this study describes an Mtb specific factor that participates in the induction of iBALT formation during TB by directly modulating cytokine and chemokine production in host cells.

Accuracy of Two Point-of-Care Tests for Rapid Diagnosis of Bovine Tuberculosis at Animal Level using Non-Invasive Specimens.

Bovine tuberculosis (BTB) testing in cattle requires a significant investment of time, equipment, and labor. Novel, rapid, cheaper and accurate methods are needed. The Alere Determine TB lipoarabinomannan antigen (LAM-test) is a World Health Organization-endorsed point-of-care urine test designed to detect active TB disease in humans. The Lionex Animal TB Rapid Test (Lionex-test) is a novel animal specific TB diagnostic blood test. An animal level analysis was performed using urine (n = 141) and milk (n = 63) samples from depopulated BTB-suspected cattle to test the accuracy of the LAM-test when compared to results of positive TB detection by any routine BTB tests (BOVIGAM, necropsy, histology, culture, PCR) that are regularly performed by the United States Department of Agriculture (USDA). The agreement between the urine LAM-test and USDA standard tests were poor at varying testing time points. The same milk samples did not elicit statistically significant agreement with the Lionex-test, although positive trends were present. Hence, we cannot recommend the LAM-test as a valid BTB diagnostic test in cattle using either urine or milk. The Lionex-test's production of positive trends using milk samples suggests larger sample sizes may validate the Lionex-test in accurately diagnosing BTB in cattle using milk samples, potentially providing a quick and reliable field test for BTB.

Evaluation of the tuberculosis culture color plate test for rapid detection of drug susceptible and drug-resistant Mycobacterium tuberculosis in a resource-limited setting, Addis Ababa, Ethiopia.

Timely diagnosis of tuberculosis (TB) is limited in Ethiopia. We evaluated the performance of a low technology, thin layer agar, Mycobacterium tuberculosis (M.tb) culture color plate (TB-CX) test with concurrent drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), and pyrazinamide (PZA) directly from sputum specimens. Patients undergoing examination for TB and multidrug-resistant (MDR)-TB were enrolled in Addis Ababa, Ethiopia from March 2016 to February 2017. All subjects received a GeneXpert MTB/RIF PCR test. TB-CX test results were compared to reference Löwenstein-Jensen (LJ) culture for M.tb detection and DST for susceptibility to INH and RIF. Kappa statistic was applied to test agreement between results for TB-CX test and the reference methods, a cut-off Kappa value of 0.75 was considered as high level of agreements. A total of 137 participants were analyzed: 88 (64%) were new TB cases, 49 (36%) were re-treatment cases. The TB-CX test detected M.tb and DST in an average of 13 days compared to 50 days for the conventional DST result. The sensitivity and specificity of the TB-CX test for detecting M.tb were 94% and 98%, respectively (concordance, 96%; kappa 0.91). The sensitivity of the TB-CX test to detect drug resistance to INH, RIF, and MDR-TB was 91%, 100%, and 90% respectively. The specificity of the TB-CX test for detecting INH, RIF, and MDR-TB was 94%, 40%, and 94% respectively. Overall agreement between TB-CX test and LJ DST for detection of MDR-TB was 93%. The TB-CX test showed strong agreement with the GeneXpert test for detecting M.tb (89%, kappa 0.76) but low agreement for the detection of RIF resistance (57%, kappa 0.28). The TB-CX test was found to be a good alternative method for screening of TB and selective drug resistant-TB in a timely and cost-efficient manner.

The Lung Mucosa Environment in the Elderly Increases Host Susceptibility to Mycobacterium tuberculosis Infection.

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.

Modifications of Pseudomonas aeruginosa cell envelope in the cystic fibrosis airway alters interactions with immune cells.

Pseudomonas aeruginosa is a ubiquitous environmental organism and an opportunistic pathogen that causes chronic lung infections in the airways of cystic fibrosis (CF) patients as well as other immune-compromised individuals. During infection, P. aeruginosa enters the terminal bronchioles and alveoli and comes into contact with alveolar lining fluid (ALF), which contains homeostatic and antimicrobial hydrolytic activities, termed hydrolases. These hydrolases comprise an array of lipases, glycosidases, and proteases and thus, they have the potential to modify lipids, carbohydrates and proteins on the surface of invading microbes. Here we show that hydrolase levels between human ALF from healthy and CF patients differ. CF-ALF influences the P. aeruginosa cell wall by reducing the content of one of its major polysaccharides, Psl. This CF-ALF induced Psl reduction does not alter initial bacterial attachment to surfaces but reduces biofilm formation. Importantly, exposure of P. aeruginosa to CF-ALF drives the activation of neutrophils and triggers their oxidative response; thus, defining human CF-ALF as a new innate defense mechanism to control P. aeruginosa infection, but at the same time potentially adding to the chronic inflammatory state of the lung in CF patients.

Mycobacterium tuberculosis Cell Wall Fragments Released upon Bacterial Contact with the Human Lung Mucosa Alter the Neutrophil Response to Infection.

In 2016, the World Health Organization reported that one person dies of tuberculosis (TB) every 21 s. A host environment that Mycobacterium tuberculosis (M.tb) finds during its route of infection is the lung mucosa bathing the alveolar space located in the deepest regions of the lungs. We published that human lung mucosa, or alveolar lining fluid (ALF), contains an array of hydrolytic enzymes that can significantly alter the M.tb surface during infection by cleaving off parts of its cell wall. This interaction results in two different outcomes: modifications on the M.tb cell wall surface and release of M.tb cell wall fragments into the environment. Typically, one of the first host immune cells at the site of M.tb infection is the neutrophil. Neutrophils can mount an extracellular and intracellular innate immune response to M.tb during infection. We hypothesized that exposure of neutrophils to ALF-induced M.tb released cell wall fragments would prime neutrophils to control M.tb infection better. Our results show that ALF fragments activate neutrophils leading to an increased production of inflammatory cytokines and oxidative radicals. However, neutrophil exposure to these fragments reduces production of chemoattractants (i.e., interleukin-8), and degranulation, with the subsequent reduction of myeloperoxidase release, and does not induce cytotoxicity. Unexpectedly, these ALF fragment-derived modulations in neutrophil activity do not further, either positively or negatively, contribute to the intracellular control of M.tb growth during infection. However, secreted products from neutrophils primed with ALF fragments are capable of regulating the activity of resting macrophages. These results indicate that ALF-induced M.tb fragments could further contribute to the control of M.tb growth and local killing by resident neutrophils by switching on the total oxidative response and limiting migration of neutrophils to the infection site.

Alveolar Epithelial Cells in Mycobacterium tuberculosis Infection: Active Players or Innocent Bystanders?

Tuberculosis (TB) is a disease that kills one person every 18 s. TB remains a global threat due to the emergence of drug-resistant Mycobacterium tuberculosis (M.tb) strains and the lack of an efficient vaccine. The ability of M.tb to persist in latency, evade recognition following seroconversion, and establish resistance in vulnerable populations warrants closer examination. Past and current research has primarily focused on examination of the role of alveolar macrophages and dendritic cells during M.tb infection, which are critical in the establishment of the host response during infection. However, emerging evidence indicates that the alveolar epithelium is a harbor for M.tb and critical during progression to active disease. Here we evaluate the relatively unexplored role of the alveolar epithelium as a reservoir and also its capacity to secrete soluble mediators upon M.tb exposure, which influence the extent of infection. We further discuss how the M.tb-alveolar epithelium interaction instigates cell-to-cell crosstalk that regulates the immune balance between a proinflammatory and an immunoregulatory state, thereby prohibiting or allowing the establishment of infection. We propose that consideration of alveolar epithelia provides a more comprehensive understanding of the lung environment in vivo in the context of host defense against M.tb.

Lung Mucosa Lining Fluid Modification of Mycobacterium tuberculosis to Reprogram Human Neutrophil Killing Mechanisms.

We have shown that human alveolar lining fluid (ALF) contains homeostatic hydrolases capable of altering the Mycobacterium tuberculosis cell wall and subsequently its interaction with human macrophages. Neutrophils are also an integral part of the host immune response to M. tuberculosis infection. Here we show that the human lung mucosa influences M. tuberculosis interaction with neutrophils, enhancing the intracellular killing of ALF-exposed M. tuberculosis and up-regulating the expression of tumor necrosis factor and interleukin 8. In contrast, ALF-exposed M. tuberculosis does not induce neutrophil apoptosis or necrosis, degranulation, or release of extracellular traps, and it decreases the oxidative response. These results suggest an important role for the human alveolar mucosa: increasing the innate capacity of the neutrophil to recognize and kill M. tuberculosis by favoring the use of intracellular mechanisms, while at the same time limiting neutrophil extracellular inflammatory responses to minimize their associated tissue damage.