Who benefits from granulomas, mycobacteria or host?

By investigating host-pathogen interactions in zebrafish using intravital imaging, Davis and Ramakrishnan (2009) provide evidence that aggregates of immune cells known as granulomas, long thought to constrain mycobacterial infection, may instead facilitate its spread.

Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium.

The human airway epithelium is the initial site of SARS-CoV-2 infection. We used flow cytometry and single cell RNA-sequencing to understand how the heterogeneity of this diverse cell population contributes to elements of viral tropism and pathogenesis, antiviral immunity, and treatment response to remdesivir. We found that, while a variety of epithelial cell types are susceptible to infection, ciliated cells are the predominant cell target of SARS-CoV-2. The host protease TMPRSS2 was required for infection of these cells. Importantly, remdesivir treatment effectively inhibited viral replication across cell types, and blunted hyperinflammatory responses. Induction of interferon responses within infected cells was rare and there was significant heterogeneity in the antiviral gene signatures, varying with the burden of infection in each cell. We also found that heavily infected secretory cells expressed abundant IL-6, a potential mediator of COVID-19 pathogenesis.

Cutting Edge: Mouse SARS-CoV-2 Epitope Reveals Infection and Vaccine-Elicited CD8 T Cell Responses.

The magnitude of SARS-CoV-2-specific T cell responses correlates inversely with human disease severity, suggesting T cell involvement in primary control. Whereas many COVID-19 vaccines focus on establishing humoral immunity to viral spike protein, vaccine-elicited T cell immunity may bolster durable protection or cross-reactivity with viral variants. To better enable mechanistic and vaccination studies in mice, we identified a dominant CD8 T cell SARS-CoV-2 nucleoprotein epitope. Infection of human ACE2 transgenic mice with SARS-CoV-2 elicited robust responses to H2-Db/N219-227, and 40% of HLA-A*02+ COVID-19 PBMC samples isolated from hospitalized patients responded to this peptide in culture. In mice, i.m. prime-boost nucleoprotein vaccination with heterologous vectors favored systemic CD8 T cell responses, whereas intranasal boosting favored respiratory immunity. In contrast, a single i.v. immunization with recombinant adenovirus established robust CD8 T cell memory both systemically and in the respiratory mucosa.

Cutting Edge: Nucleocapsid Vaccine Elicits Spike-Independent SARS-CoV-2 Protective Immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing Abs target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with a human adenovirus type 5 vector expressing the SARS-CoV-2 nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and K18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral Ags in SARS-CoV-2 vaccines, even if they are not a target of neutralizing Abs, to broaden epitope coverage and immune effector mechanisms.

Immune Profiling to Determine Early Disease Trajectories Associated With Coronavirus Disease 2019 Mortality Rate: A Substudy from the ACTT-1 Trial.

Coronavirus disease 2019 (COVID-19) outcomes are linked to host immune responses and may be affected by antiviral therapy. We investigated antibody and cytokine responses in ACTT-1 study participants enrolled at our center. We studied serum specimens from 19 hospitalized adults with COVID-19 randomized to treatment with remdesivir or placebo. We assessed severe acute respiratory syndrome coronavirus 2 antibody responses and identified cytokine signatures, using hierarchical clustering. We identified no clear immunologic trends attributable to remdesivir treatment. Seven participants were initially seronegative at study enrollment, and all 4 deaths occurred in this group with more recent symptom onset. We identified 3 dominant cytokine signatures, demonstrating different disease trajectories.

Cerebrospinal Fluid Bacillary Load by Xpert MTB/RIF Ultra Polymerase Chain Reaction Cycle Threshold Value Predicts 2-Week Mortality in Human Immunodeficiency Virus-Associated Tuberculous Meningitis.

BACKGROUND: The World Health Organization recommends GeneXpert MTB/RIF Ultra (Xpert Ultra), a fully automated polymerase chain reaction (PCR) assay, as the initial tuberculous meningitis (TBM) diagnostic test. The assay's PCR cycle threshold (Ct) values represent the number of PCR cycles required for probe signal to be detected (low Ct value = high bacillary load) and may approximate tuberculosis (TB) bacillary load. We measured the relationship between cerebrospinal fluid (CSF) TB bacillary load with mortality. METHODS: We prospectively enrolled 102 human immunodeficiency virus (HIV)-positive Ugandans with probable or definite TBM from April 2015 to August 2019. Xpert Ultra Ct tertiles and semi-quantitative categories were separately analyzed as predictors of 2-week mortality. We investigated associations between Ct and baseline clinical and CSF parameters. RESULTS: Subjects with Ct values in the low tertile (ie, high bacillary load) had 57% 2-week mortality-worse than the intermediate (17%) and high (25%) Ct tertiles and Xpert Ultra-negative (30%) probable TBM cases (P = .01). In contrast, the reported semi-quantitative Xpert Ultra categorization was less precise; with the medium to low category trending toward worse 2-week survival (42%) compared with very low (28%), trace (26%), and negative (30%) categories (P = .48). Ct tertile was significantly associated with baseline CSF lactate (P = .03). CONCLUSIONS: High CSF TB bacillary load, as measured by Xpert Ultra Ct tertile, is associated with an almost 2-fold higher 2-week mortality in HIV-associated TBM and is a better predictor than the reported Xpert Ultra semi-quantitative category. Xpert Ultra Ct values could identify TBM patients at increased risk of death who may benefit from enhanced supportive care.

SARS-CoV-2 neutralization and serology testing of COVID-19 convalescent plasma from donors with nonsevere disease.

BACKGROUND: The transfer of passive immunity with convalescent plasma is a promising strategy for treatment and prevention of COVID-19, but donors with a history of nonsevere disease are serologically heterogenous. The relationship between SARS-Cov-2 antigen-binding activity and neutralization activity in this population of donors has not been defined. STUDY DESIGN AND METHODS: Convalescent plasma units from 47 individuals with a history of nonsevere COVID-19 were assessed for antigen-binding activity of using three clinical diagnostic serology assays (Beckman, DiaSorin, and Roche) with different SARS-CoV-2 targets. These results were compared with functional neutralization activity using a fluorescent reporter strain of SARS-CoV-2 in a microwell assay. RESULTS: Positive correlations of varying strength (Spearman r = 0.37-0.52) between antigen binding and viral neutralization were identified. Donors age 48 to 75 years had the highest neutralization activity. Units in the highest tertile of binding activity for each assay were enriched (75%-82%) for those with the highest levels of neutralization. CONCLUSION: The strength of the relationship between antigen-binding activity and neutralization varies depending on the clinical assay used. Units in the highest tertile of binding activity for each assay are predominantly comprised of those with the greatest neutralization activity.

A eulogy for Dr Francisco Miguel Marty Forero.

As some of those who were lucky enough to have been mentored by Dr Francisco Marty in transplant infectious diseases, we stand with the larger medical community in mourning his untimely death and in commemorating him as a uniquely exceptional and talented physician, investigator, teacher, mentor, friend, artist, and human being.

Use of triazoles for the treatment of invasive aspergillosis: A three-year cohort analysis.

In a 3-year cohort study of adult patients with proven or probable IA, fewer patients initially treated with isavuconazole experienced adverse events compared with voriconazole, but more patients required a change in therapy due to lack of clinical efficacy.

Risk of Latent Tuberculosis Reactivation After Hematopoietic cell Transplantation.

There were no cases of tuberculosis in a cohort of 2531 patients who underwent hematopoietic cell transplantation from 2010 to 2015 after 7323 person-years of follow up (95% confidence interval [CI], 0.0-0.05 cases/100 person-years), including 29 (1.15%) patients with untreated latent tuberculosis after 89 person-years of follow-up (95% CI, 0.0-4.06 cases/100 person-years).

CD4+ T cell-dependent IFN-γ production by CD8+ effector T cells in Mycobacterium tuberculosis infection.

Both CD4+ and CD8+ T cells contribute to immunity to tuberculosis, and both can produce the essential effector cytokine IFN-γ. However, the precise role and relative contribution of each cell type to in vivo IFN-γ production are incompletely understood. To identify and quantitate the cells that produce IFN-γ at the site of Mycobacterium tuberculosis infection in mice, we used direct intracellular cytokine staining ex vivo without restimulation. We found that CD4+ and CD8+ cells were predominantly responsible for production of this cytokine in vivo, and we observed a remarkable linear correlation between the fraction of CD4+ cells and the fraction of CD8+ cells producing IFN-γ in the lungs. In the absence of CD4+ cells, a reduced fraction of CD8+ cells was actively producing IFN-γ in vivo, suggesting that CD4+ effector cells are continually required for optimal IFN-γ production by CD8+ effector cells. Accordingly, when infected mice were treated i.v. with an MHC-II-restricted M. tuberculosis epitope peptide to stimulate CD4+ cells in vivo, we observed rapid activation of both CD4+ and CD8+ cells in the lungs. Indirect activation of CD8+ cells was dependent on the presence of CD4+ cells but independent of IFN-g responsiveness of the CD8+ cells. These data provide evidence that CD4+ cell deficiency impairs IFN-γ production by CD8+ effector cells and that ongoing cross-talk between distinct effector T cell types in the lungs may contribute to a protective immune response against M. tuberculosis. Conversely, defects in these interactions may contribute to susceptibility to tuberculosis and other infections.

Adrenal gland macrophages regulate glucocorticoid production through Trem2 and TGFß.

Glucocorticoid synthesis by adrenal glands (AG) is regulated by the hypothalamic-pituitary-adrenal axis (HPA-axis) to facilitate stress responses when the host is exposed to stimuli. Recent studies have implicated macrophages (MФ) as potential steroidogenic regulators, but the molecular mechanisms by which AG MФ exert such influence remain unclear. In this study, we investigated the role of AG MФ in response to cold challenge or atherosclerotic inflammation as physiologic models of acute or chronic stress. Utilizing single-cell RNA sequencing, we observed dynamic AG MФ polarization toward classical activation and lipid-associated phenotypes following acute or chronic stimulation. Among the transcriptional alterations induced in MФ, Triggering Receptor Expressed on Myeloid (Trem2) was highlighted due to its dramatic upregulation following stress. Conditional deletion of MФ Trem2 revealed a protective role for Trem2 in stress responses. Mechanistically, Trem2 deletion led to increased AG MФ death, abolished the TGFß-producing capacity of AG MФ, and resulted in enhanced glucocorticoid production. In addition, enhanced glucocorticoid production was replicated by blockade of TGFß signaling. Together, these observations suggest that AG MФ restrict steroidogenesis through Trem2 and TGFß, which opens potential avenues for immunotherapeutic interventions targeting the innate immune system to resolve stress-related disorders.

Suboptimal activation of antigen-specific CD4+ effector cells enables persistence of M. tuberculosis in vivo.

Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-γ without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-γ production. During the early phase of infection, ∼10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.

Impaired fitness of Mycobacterium africanum despite secretion of ESAT-6.

BACKGROUND: When compared with Mycobacterium tuberculosis, individuals that live in the same household as an active case of smear-positive pulmonary tuberculosis exposed to M. africanum progress less frequently to active disease within 2 years. A putative ESX-1 secretion apparatus member, Rv3879c, is mutated in M. africanum, and individuals infected with M. africanum less frequently demonstrate T-cell responses to the ESX-1-secreted virulence factor ESAT-6 than those infected with M. tuberculosis. We hypothesized that less frequent progression is caused by impaired secretion of ESAT-6. METHODS: We analyzed in vivo growth and in vitro secretion of ESAT-6 and CFP-10, comparing M. tuberculosis to M. africanum and a strain of M. africanum complemented with M. tuberculosis Rv3879c. RESULTS: ESAT-6 and CFP-10 secretion were similar for all strains, although these were enriched in M. africanum cell lysates, suggesting a modest ESX-1 secretion defect unrelated to the Rv3879c mutation. In mice, M. africanum demonstrated smaller bacterial population sizes than M. tuberculosis but similar numbers and frequencies of ESAT-6-responsive T cells in the lungs. CONCLUSIONS: These results confirm impaired fitness of M. africanum in vivo and indicate that Rv3879c is not required for secretion of ESAT-6 or for its presentation as an antigen to T cells in vivo.

A new hope; the M. tuberculosis strikes back.

The development of new tools to combat TB is counterbalanced by the discovery of previously unknown biological mechanisms used by M. tuberculosis to evade eradication. Two new studies offer both new hope, in a promising ribosome-targeting TB therapy, as well as a new challenge to overcome, in antibiotic resilience.

TB granuloma: CD30 co-stimulation for CD4+ T cell co-operation.

Tuberculosis granuloma T cells express an array of mediators including the CD30 co-stimulatory receptor and its ligand, CD153. CD4 T effector cells require signals through CD30, potentially provided co-operatively by other T cells, to completely differentiate and protect against disease (Foreman et al., 2023. J. Exp. Med.https://doi.org/10.1084/jem.20222090).

Recently activated CD4 T cells in tuberculosis express OX40 as a target for host-directed immunotherapy.

After Mycobacterium tuberculosis (Mtb) infection, many effector T cells traffic to the lungs, but few become activated. Here we use an antigen receptor reporter mouse (Nur77-GFP) to identify recently activated CD4 T cells in the lungs. These Nur77-GFPHI cells contain expanded TCR clonotypes, have elevated expression of co-stimulatory genes such as Tnfrsf4/OX40, and are functionally more protective than Nur77-GFPLO cells. By contrast, Nur77-GFPLO cells express markers of terminal exhaustion and cytotoxicity, and the trafficking receptor S1pr5, associated with vascular localization. A short course of immunotherapy targeting OX40+ cells transiently expands CD4 T cell numbers and shifts their phenotype towards parenchymal protective cells. Moreover, OX40 agonist immunotherapy decreases the lung bacterial burden and extends host survival, offering an additive benefit to antibiotics. CD4 T cells from the cerebrospinal fluid of humans with HIV-associated tuberculous meningitis commonly express surface OX40 protein, while CD8 T cells do not. Our data thus propose OX40 as a marker of recently activated CD4 T cells at the infection site and a potential target for immunotherapy in tuberculosis.

Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis.

Atherosclerosis is driven by the expansion of cholesterol-loaded 'foamy' macrophages in the arterial intima. Factors regulating foamy macrophage differentiation and survival in plaque remain poorly understood. Here we show, using trajectory analysis of integrated single-cell RNA sequencing data and a genome-wide CRISPR screen, that triggering receptor expressed on myeloid cells 2 (Trem2) is associated with foamy macrophage specification. Loss of Trem2 led to a reduced ability of foamy macrophages to take up oxidized low-density lipoprotein (oxLDL). Myeloid-specific deletion of Trem2 showed an attenuation of plaque progression, even when targeted in established atherosclerotic lesions, and was independent of changes in circulating cytokines, monocyte recruitment or cholesterol levels. Mechanistically, we link Trem2-deficient macrophages with a failure to upregulate cholesterol efflux molecules, resulting in impaired proliferation and survival. Overall, we identify Trem2 as a regulator of foamy macrophage differentiation and atherosclerotic plaque growth and as a putative therapeutic target for atherosclerosis.

A New Strategy to Prevent "Deja vUTI".

Deja vu is the sensation of having a recurrent experience. Within a year after an initial urinary tract infection (UTI), up to 30% of women will experience a recurrence of UTI symptoms.1 A smaller group of women (1%) may experience frequent recurrence with six or more episodes in a year. Recurrent UTI, or "deja vUTI," is a challenging problem that still lacks a cohesive pathophysiological mechanism, and it is currently addressed with a wide range of clinical practices often based on experience rather than on high-quality evidence.