Imaging the Architecture of Granulomas Induced by Mycobacterium tuberculosis Infection with Single-molecule Fluorescence In Situ Hybridization.

Granulomas are an important hallmark of Mycobacterium tuberculosis infection. They are organized and dynamic structures created when immune cells assemble around the sites of infection in the lungs that locally restrict M. tuberculosis growth and the host's inflammatory responses. The cellular architecture of granulomas is traditionally studied by immunofluorescence labeling of surface markers on the host cells. However, very few Abs are available for model animals used in tuberculosis research, such as nonhuman primates and rabbits, and secreted immunological markers such as cytokines cannot be imaged in situ using Abs. Furthermore, traditional phenotypic surface markers do not provide sufficient resolution for the detection of the many subtypes and differentiation states of immune cells. Using single-molecule fluorescence in situ hybridization (smFISH) and its derivatives, amplified smFISH and iterative smFISH, we developed a platform for imaging mRNAs encoding immune markers in rabbit and macaque tuberculosis granulomas. Multiplexed imaging for several mRNA and protein markers was followed by quantitative measurement of the expression of these markers in single cells. An analysis of the combinatorial expressions of these markers allowed us to classify the cells into several subtypes, and to chart their densities within granulomas. For one mRNA target, hypoxia-inducible factor-1α, we imaged its mRNA and protein in the same cells, demonstrating the specificity of the probes. This method paves the way for defining granular differentiation states and cell subtypes from transcriptomic data, identifying key mRNA markers for these cell subtypes, and then locating the cells in the spatial context of granulomas.

Imaging and quantification of human and viral circular RNAs.

We present a robust approach for cellular detection, imaging, localization, and quantification of human and viral encoded circular RNAs (circRNA) using amplified fluorescence in situ hybridization (ampFISH). In this procedure, a pair of hairpin probes bind next to each other at contiguous stretches of sequence and then undergo a conformational reorganization which initiates a target-dependent hybridization chain reaction (HCR) resulting in deposition of an amplified fluorescent signal at the site. By harnessing the capabilities of both ampFISH and single-molecule FISH (smFISH), we selectively identified and imaged circular RNAs and their linear counterparts derived from the human genome, SARS-CoV-2 (an RNA virus), and human cytomegalovirus (HCMV, a DNA virus). Computational image processing facilitated accurate quantification of circular RNA molecules in individual cells. The specificity of ampFISH for circular RNA detection was confirmed through an in situ RNase R treatment that selectively degrades linear RNAs without impacting circular RNAs. The effectiveness of circular RNA detection was further validated by using ampFISH probes with mismatches and probe pairs that do not bind to the continuous sequence in their target RNAs but instead bind at segregated sites. An additional specificity test involved probes against the negative strands of the circular RNA sequence, absent in the cell. Importantly, our technique allows simultaneous detection of circular RNAs and their linear counterparts within the same cell with single molecule sensitivity, enabling explorations of circular RNA biogenesis, subcellular localization, and functions.

Eicosanoid regulation of debris-stimulated metastasis.

Cancer therapy reduces tumor burden via tumor cell death ("debris"), which can accelerate tumor progression via the failure of inflammation resolution. Thus, there is an urgent need to develop treatment modalities that stimulate the clearance or resolution of inflammation-associated debris. Here, we demonstrate that chemotherapy-generated debris stimulates metastasis by up-regulating soluble epoxide hydrolase (sEH) and the prostaglandin E2 receptor 4 (EP4). Therapy-induced tumor cell debris triggers a storm of proinflammatory and proangiogenic eicosanoid-driven cytokines. Thus, targeting a single eicosanoid or cytokine is unlikely to prevent chemotherapy-induced metastasis. Pharmacological abrogation of both sEH and EP4 eicosanoid pathways prevents hepato-pancreatic tumor growth and liver metastasis by promoting macrophage phagocytosis of debris and counterregulating a protumorigenic eicosanoid and cytokine storm. Therefore, stimulating the clearance of tumor cell debris via combined sEH and EP4 inhibition is an approach to prevent debris-stimulated metastasis and tumor growth.

Immunopathology of Pulmonary Mycobacterium tuberculosis Infection in a Humanized Mouse Model.

Despite the availability of antibiotic therapy, tuberculosis (TB) is prevailing as a leading killer among human infectious diseases, which highlights the need for better intervention strategies to control TB. Several animal model systems, including mice, guinea pigs, rabbits, and non-human primates have been developed and explored to understand TB pathogenesis. Although each of these models contributes to our current understanding of host-Mycobacterium tuberculosis (Mtb) interactions, none of these models fully recapitulate the pathological spectrum of clinical TB seen in human patients. Recently, humanized mouse models are being developed to improvise the limitations associated with the standard mouse model of TB, including lack of necrotic caseation of granulomas, a pathological hallmark of TB in humans. However, the spatial immunopathology of pulmonary TB in humanized mice is not fully understood. In this study, using a novel humanized mouse model, we evaluated the spatial immunopathology of pulmonary Mtb infection with a low-dose inoculum. Humanized NOD/LtSscidIL2Rγ null mice containing human fetal liver, thymus, and hematopoietic CD34+ cells and treated with human cytokines were aerosol challenged to implant <50 pathogenic Mtb (low dose) in the lungs. At 2 and 4 weeks post infection, the tissue bacterial load, disease pathology, and spatial immunohistology were determined in the lungs, liver, spleen, and adipose tissue using bacteriological, histopathological, and immunohistochemical techniques. The results indicate that implantation of <50 bacteria can establish a progressive disease in the lungs that transmits to other tissues over time. The disease pathology in organs correspondingly increased with the bacterial load. A distinct spatial distribution of T cells, macrophages, and natural killer cells were noted in the lung granulomas. The kinetics of spatial immune cell distribution were consistent with the disease pathology in the lungs. Thus, the novel humanized model recapitulates several key features of human pulmonary TB granulomatous response and can be a useful preclinical tool to evaluate potential anti-TB drugs and vaccines.

COVID-19 and cancer: start the resolution!

Coronavirus disease 2019 (COVID-19) due to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been an ongoing pandemic causing significant morbidity and mortality worldwide. The "cytokine storm" is a critical driving force in severe COVID-19 cases, leading to hyperinflammation, multi-system organ failure, and death. A paradigm shift is emerging in our understanding of the resolution of inflammation from a passive course to an active biochemical process driven by endogenous specialized pro-resolving mediators (SPMs), such as resolvins, protectins, lipoxins, and maresins. SPMs stimulate macrophage-mediated debris clearance and counter pro-inflammatory cytokine production, a process collectively termed as the "resolution of inflammation." Hyperinflammation is not unique to COVID-19 and also occurs in neoplastic conditions, putting individuals with underlying health conditions such as cancer at elevated risk of severe SARS-CoV-2 infection. Despite approaches to block systemic inflammation, there are no current therapies designed to stimulate the resolution of inflammation in patients with COVID-19 or cancer. A non-immunosuppressive therapeutic approach that reduces the cytokine storm in patients with COVID-19 and cancer is urgently needed. SPMs are potent immunoresolvent and organ-protective lipid autacoids that stimulate the resolution of inflammation, facilitate clearance of infections, reduce thrombus burden, and promote a return to tissue homeostasis. Targeting endogenous lipid mediators, such as SPMs, offers an entirely novel approach to control SARS-CoV-2 infection and cancer by increasing the body's natural reserve of pro-resolving mediators without overt toxicity or immunosuppression.

Simplified urine-based method to detect rifampin underexposure in adults with tuberculosis: a prospective diagnostic accuracy study.

Variable pharmacokinetics of rifampin in tuberculosis (TB) treatment can lead to poor outcomes. Urine spectrophotometry is simpler and more accessible than recommended serum-based drug monitoring, but its optimal efficacy in predicting serum rifampin underexposure in adults with TB remains uncertain. Adult TB patients in New Jersey and Virginia receiving rifampin-containing regimens were enrolled. Serum and urine samples were collected over 24 h. Rifampin serum concentrations were measured using validated liquid chromatography-tandem mass spectrometry, and total exposure (area under the concentration-time curve) over 24 h (AUC0-24) was determined through noncompartmental analysis. The Sunahara method was used to extract total rifamycins, and rifampin urine excretion was measured by spectrophotometry. An analysis of 58 eligible participants, including 15 (26%) with type 2 diabetes mellitus, demonstrated that urine spectrophotometry accurately identified subtarget rifampin AUC0-24 at 0-4, 0-8, and 0-24 h. The area under the receiver operator characteristic curve (AUC ROC) values were 0.80 (95% CI 0.67-0.90), 0.84 (95% CI 0.72-0.94), and 0.83 (95% CI 0.72-0.93), respectively. These values were comparable to the AUC ROC of 2 h serum concentrations commonly used for therapeutic monitoring (0.82 [95% CI 0.71-0.92], P = 0.6). Diabetes status did not significantly affect the AUC ROCs for urine in predicting subtarget rifampin serum exposure (P = 0.67-0.92). Spectrophotometric measurement of urine rifampin excretion within the first 4 or 8 h after dosing is a simple and cost-effective test that accurately predicts rifampin underexposure. This test provides critical information for optimizing tuberculosis treatment outcomes by facilitating appropriate dose adjustments.

Critical Determinants of Cytokine Storm and Type I Interferon Response in COVID-19 Pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a rapidly evolving pandemic worldwide with at least 68 million COVID-19-positive cases and a mortality rate of about 2.2%, as of 10 December 2020. About 20% of COVID-19 patients exhibit moderate to severe symptoms. Severe COVID-19 manifests as acute respiratory distress syndrome (ARDS) with elevated plasma proinflammatory cytokines, including interleukin 1ß (IL-1ß), IL-6, tumor necrosis factor α (TNF-α), C-X-C motif chemokine ligand 10 (CXCL10/IP10), macrophage inflammatory protein 1 alpha (MIP-1α), and chemokine (C-C motif) ligand 2 (CCL2), with low levels of interferon type I (IFN-I) in the early stage and elevated levels of IFN-I during the advanced stage of COVID-19. Most of the severe and critically ill COVID-19 patients have had preexisting comorbidities, including hypertension, diabetes, cardiovascular diseases, and respiratory diseases. These conditions are known to perturb the levels of cytokines, chemokines, and angiotensin-converting enzyme 2 (ACE2), an essential receptor involved in SARS-CoV-2 entry into the host cells. ACE2 downregulation during SARS-CoV-2 infection activates the angiotensin II/angiotensin receptor (AT1R)-mediated hypercytokinemia and hyperinflammatory syndrome. However, several SARS-CoV-2 proteins, including open reading frame 3b (ORF3b), ORF6, ORF7, ORF8, and the nucleocapsid (N) protein, can inhibit IFN type I and II (IFN-I and -II) production. Thus, hyperinflammation, in combination with the lack of IFN responses against SARS-CoV-2 early on during infection, makes the patients succumb rapidly to COVID-19. Therefore, therapeutic approaches involving anti-cytokine/anti-cytokine-signaling and IFN therapy would favor the disease prognosis in COVID-19. This review describes critical host and viral factors underpinning the inflammatory "cytokine storm" induction and IFN antagonism during COVID-19 pathogenesis. Therapeutic approaches to reduce hyperinflammation and their limitations are also discussed.

Different modalities of host cell death and their impact on Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (Mtb) is the pathogen that causes tuberculosis (TB), a leading infectious disease of humans worldwide. One of the main histopathological hallmarks of TB is the formation of granulomas comprised of elaborately organized aggregates of immune cells containing the pathogen. Dissemination of Mtb from infected cells in the granulomas due to host and mycobacterial factors induces multiple cell death modalities in infected cells. Based on molecular mechanism, morphological characteristics, and signal dependency, there are two main categories of cell death: programmed and nonprogrammed. Programmed cell death (PCD), such as apoptosis and autophagy, is associated with a protective response to Mtb by keeping the bacteria encased within dead macrophages that can be readily phagocytosed by arriving in uninfected or neighboring cells. In contrast, non-PCD necrotic cell death favors the pathogen, resulting in bacterial release into the extracellular environment. Multiple types of cell death in the PCD category, including pyroptosis, necroptosis, ferroptosis, ETosis, parthanatos, and PANoptosis, may be involved in Mtb infection. Since PCD pathways are essential for host immunity to Mtb, therapeutic compounds targeting cell death signaling pathways have been experimentally tested for TB treatment. This review summarizes different modalities of Mtb-mediated host cell deaths, the molecular mechanisms underpinning host cell death during Mtb infection, and its potential implications for host immunity. In addition, targeting host cell death pathways as potential therapeutic and preventive approaches against Mtb infection is also discussed.

Lipid-Polymer Hybrid "Particle-in-Particle" Nanostructure Gene Delivery Platform Explored for Lyophilizable DNA and mRNA COVID-19 Vaccines.

SARS-CoV-2 has led to a worldwide pandemic, catastrophically impacting public health and the global economy. Herein, a new class of lipid-modified polymer poly (ß-amino esters) (L-PBAEs) is developed via enzyme-catalyzed esterification and further formulation of the L-PBAEs with poly(d,l-lactide-coglycolide)-b-poly(ethylene glycol) (PLGA-PEG) leads to self-assembly into a "particle-in-particle" (PNP) nanostructure for gene delivery. Out of 24 PNP candidates, the top-performing PNP/C12-PBAE nanoparticles efficiently deliver both DNA and mRNA in vitro and in vivo, presenting enhanced transfection efficacy, sustained gene release behavior, and excellent stability for at least 12 months of storage at -20 °C after lyophilization without loss of transfection efficacy. Encapsulated with spike encoded plasmid DNA and mRNA, the lipid-modified polymeric PNP COVID-19 vaccines successfully elicit spike-specific antibodies and Th1-biased T cell immune responses in immunized mice even after 12 months of lyophilized storage at -20 °C. This newly developed lipid-polymer hybrid PNP nanoparticle system demonstrates a new strategy for both plasmid DNA and mRNA delivery with the capability of long-term lyophilized storage.

SARS-CoV-2, SARS-CoV, and MERS-CoV encode circular RNAs of spliceosome-independent origin.

Circular RNAs (circRNAs) are a newly recognized component of the transcriptome with critical roles in autoimmune diseases and viral pathogenesis. To address the importance of circRNA in RNA viral transcriptome, we systematically identified and characterized circRNAs encoded by the RNA genomes of betacoronaviruses using both bioinformatical and experimental approaches. We predicted 351, 224, and 2764 circRNAs derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus, respectively. We experimentally identified 75 potential SARS-CoV-2 circRNAs from RNA samples extracted from SARS-CoV-2-infected Vero E6 cells. A systematic comparison of viral and host circRNA features, including abundance, strand preference, length distribution, circular exon numbers, and breakpoint sequences, demonstrated that coronavirus-derived circRNAs had a spliceosome-independent origin. We further showed that back-splice junctions (BSJs) captured by inverse reverse-transcription polymerase chain reaction have different level of resistance to RNase R. Through northern blotting with a BSJ-spanning probe targeting N gene, we identified three RNase R-resistant bands that represent SARS-CoV-2 circRNAs that are detected cytoplasmic by single-molecule and amplified fluorescence in situ hybridization assays. Lastly, analyses of 169 sequenced BSJs showed that both back-splice and forward-splice junctions were flanked by homologous and reverse complementary sequences, including but not limited to the canonical transcriptional regulatory sequences. Our findings highlight circRNAs as an important component of the coronavirus transcriptome, offer important evaluation of bioinformatic tools in the analysis of circRNAs from an RNA genome, and shed light on the mechanism of discontinuous RNA synthesis.

Ultrasensitive Detection of Multidrug-Resistant Mycobacterium tuberculosis Using SuperSelective Primer-Based Real-Time PCR Assays.

The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant Mycobacterium tuberculosis is critical to developing fully drug-resistant tuberculosis cases. The currently available molecular techniques may detect one copy of mutant bacterial genomic DNA in the presence of about 1-1000 copies of wild-type M. tuberculosis DNA. To improve the limit of heteroresistance detection, we developed SuperSelective primer-based real-time PCR assays, which, by their unique assay design, enable selective and exponential amplification of selected point mutations in the presence of abundant wild-type DNA. We designed SuperSelective primers to detect genetic mutations associated with M. tuberculosis resistance to the anti-tuberculosis drugs isoniazid and rifampin. We evaluated the efficiency of our assay in detecting heteroresistant M. tuberculosis strains using genomic DNA isolated from laboratory strains and clinical isolates from the sputum of tuberculosis patients. Results show that our assays detected heteroresistant mutations with a specificity of 100% in a background of up to 104 copies of wild-type M. tuberculosis genomic DNA, corresponding to a detection limit of 0.01%. Therefore, the SuperSelective primer-based RT-PCR assay is an ultrasensitive tool that can efficiently diagnose heteroresistant tuberculosis in clinical specimens and contributes to understanding the drug resistance mechanisms. This approach can improve the management of antimicrobial resistance in tuberculosis and other infectious diseases.

Inoculum size and traits of the infecting clinical strain define the protection level against Mycobacterium tuberculosis infection in a rabbit model.

Host protective immunity against pathogenic Mycobacterium tuberculosis (Mtb) infection is variable and poorly understood. Both prior Mtb infection and BCG vaccination have been reported to confer some protection against subsequent infection and/or disease. However, the immune correlates of host protection with or without BCG vaccination remain poorly understood. Similarly, the host response to concomitant infection with mixed Mtb strains is unclear. In this study, we used the rabbit model to examine the host response to various infectious doses of virulent Mtb HN878 with and without prior BCG vaccination, as well as simultaneous infection with a mixture of two Mtb clinical isolates HN878 and CDC1551. We demonstrate that both the ability of host immunity to control pulmonary Mtb infection and the protective efficacy of BCG vaccination against the progression of Mtb infection to disease is dependent on the infectious inoculum. The host response to infection with mixed Mtb strains mirrors the differential responses seen during infection with each of the strains alone. The protective response mounted against a hyperimmunogenic Mtb strain has a limited impact on the control of disease caused by a hypervirulent strain. This preclinical study will aid in predicting the success of any vaccination strategy and in optimizing TB vaccines.

Corticosteroids for COVID-19 Therapy: Potential Implications on Tuberculosis.

On 11 March 2020, the World Health Organization announced the Corona Virus Disease-2019 (COVID-19) as a global pandemic, which originated in China. At the host level, COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), affects the respiratory system, with the clinical symptoms ranging from mild to severe or critical illness that often requires hospitalization and oxygen support. There is no specific therapy for COVID-19, as is the case for any common viral disease except drugs to reduce the viral load and alleviate the inflammatory symptoms. Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb), also primarily affects the lungs and has clinical signs similar to pulmonary SARS-CoV-2 infection. Active TB is a leading killer among infectious diseases and adds to the burden of the COVID-19 pandemic worldwide. In immunocompetent individuals, primary Mtb infection can also lead to a non-progressive, asymptomatic latency. However, latent Mtb infection (LTBI) can reactivate symptomatic TB disease upon host immune-suppressing conditions. Importantly, the diagnosis and treatment of TB are hampered and admixed with COVID-19 control measures. The US-Center for Disease Control (US-CDC) recommends using antiviral drugs, Remdesivir or corticosteroid (CST), such as dexamethasone either alone or in-combination with specific recommendations for COVID-19 patients requiring hospitalization or oxygen support. However, CSTs can cause immunosuppression, besides their anti-inflammatory properties. The altered host immunity during COVID-19, combined with CST therapy, poses a significant risk for new secondary infections and/or reactivation of existing quiescent infections, such as LTBI. This review highlights CST therapy recommendations for COVID-19, various types and mechanisms of action of CSTs, the deadly combination of two respiratory infectious diseases COVID-19 and TB. It also discusses the importance of screening for LTBI to prevent TB reactivation during corticosteroid therapy for COVID-19.

Storage lipid studies in tuberculosis reveal that foam cell biogenesis is disease-specific.

Foam cells are lipid-laden macrophages that contribute to the inflammation and tissue damage associated with many chronic inflammatory disorders. Although foam cell biogenesis has been extensively studied in atherosclerosis, how these cells form during a chronic infectious disease such as tuberculosis is unknown. Here we report that, unlike the cholesterol-laden cells of atherosclerosis, foam cells in tuberculous lung lesions accumulate triglycerides. Consequently, the biogenesis of foam cells varies with the underlying disease. In vitro mechanistic studies showed that triglyceride accumulation in human macrophages infected with Mycobacterium tuberculosis is mediated by TNF receptor signaling through downstream activation of the caspase cascade and the mammalian target of rapamycin complex 1 (mTORC1). These features are distinct from the known biogenesis of atherogenic foam cells and establish a new paradigm for non-atherogenic foam cell formation. Moreover, they reveal novel targets for disease-specific pharmacological interventions against maladaptive macrophage responses.

Of tuberculosis and non-tuberculous mycobacterial infections - a comparative analysis of epidemiology, diagnosis and treatment.

Pulmonary diseases due to mycobacteria cause significant morbidity and mortality to human health. In addition to tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), recent epidemiological studies have shown the emergence of non-tuberculous mycobacteria (NTM) species in causing lung diseases in humans. Although more than 170 NTM species are present in various environmental niches, only a handful, primarily Mycobacterium avium complex and M. abscessus, have been implicated in pulmonary disease. While TB is transmitted through inhalation of aerosol droplets containing Mtb, generated by patients with symptomatic disease, NTM disease is mostly disseminated through aerosols originated from the environment. However, following inhalation, both Mtb and NTM are phagocytosed by alveolar macrophages in the lungs. Subsequently, various immune cells are recruited from the circulation to the site of infection, which leads to granuloma formation. Although the pathophysiology of TB and NTM diseases share several fundamental cellular and molecular events, the host-susceptibility to Mtb and NTM infections are different. Striking differences also exist in the disease presentation between TB and NTM cases. While NTM disease is primarily associated with bronchiectasis, this condition is rarely a predisposing factor for TB. Similarly, in Human Immunodeficiency Virus (HIV)-infected individuals, NTM disease presents as disseminated, extrapulmonary form rather than as a miliary, pulmonary disease, which is seen in Mtb infection. The diagnostic modalities for TB, including molecular diagnosis and drug-susceptibility testing (DST), are more advanced and possess a higher rate of sensitivity and specificity, compared to the tools available for NTM infections. In general, drug-sensitive TB is effectively treated with a standard multi-drug regimen containing well-defined first- and second-line antibiotics. However, the treatment of drug-resistant TB requires the additional, newer class of antibiotics in combination with or without the first and second-line drugs. In contrast, the NTM species display significant heterogeneity in their susceptibility to standard anti-TB drugs. Thus, the treatment for NTM diseases usually involves the use of macrolides and injectable aminoglycosides. Although well-established international guidelines are available, treatment of NTM disease is mostly empirical and not entirely successful. In general, the treatment duration is much longer for NTM diseases, compared to TB, and resection surgery of affected organ(s) is part of treatment for patients with NTM diseases that do not respond to the antibiotics treatment. Here, we discuss the epidemiology, diagnosis, and treatment modalities available for TB and NTM diseases of humans.

Role of the inositol pyrophosphate multikinase Kcs1 in Cryptococcus inositol metabolism.

Cryptococcus neoformans is the most common cause of deadly fungal meningitis. This fungus has a complex inositol acquisition and utilization system, and our previous studies have shown the importance of inositol utilization in cryptococcal development and virulence. However, how inositol utilization is regulated in this fungus remains unknown. In this study, we found that inositol, irrespective of the presence of glucose in the media, represses the expression of C. neoformans genes involved in inositol pyrophosphate biosynthesis, including the gene encoding inositol hexakisphosphate kinase Kcs1. Kcs1 was recently reported to regulate inositol metabolism in Saccharomyces cerevisiae and to impact virulence in C. neoformans. To examine the potential role of Kcs1 in inositol regulation in C. neoformans, we generated the kcs1Δ mutant and compared its phenotype with the wild type strain. We found that Kcs1 negatively regulates inositol uptake and catabolism in C. neoformans, but, in contrast to Kcs1 function in S. cerevisiae, does not appear to regulate inositol biosynthesis. Together, these results show that Kcs1 functions to fine-tune inositol acquisition to maintain inositol homeostasis in C. neoformans.

Macrophage migration inhibitory factor (MIF) is a critical mediator of the innate immune response to Mycobacterium tuberculosis.

Macrophage migration inhibitory factor (MIF), an innate cytokine encoded in a functionally polymorphic genetic locus, contributes to detrimental inflammation but may be crucial for controlling infection. We explored the role of variant MIF alleles in tuberculosis. In a Ugandan cohort, genetic low expressers of MIF were 2.4-times more frequently identified among patients with Mycobacterium tuberculosis (TB) bacteremia than those without. We also found mycobacteria-stimulated transcription of MIF and serum MIF levels to be correlated with MIF genotype in human macrophages and in a separate cohort of US TB patients, respectively. To determine mechanisms for MIF's protective role, we studied both aerosolized and i.v. models of mycobacterial infection and observed MIF-deficient mice to succumb more quickly with higher organism burden, increased lung pathology, and decreased innate cytokine production (TNF-α, IL-12, IL-10). MIF-deficient animals showed increased pulmonary neutrophil accumulation but preserved adaptive immune response. MIF-deficient macrophages demonstrated decreased cytokine and reactive oxygen production and impaired mycobacterial killing. Transcriptional investigation of MIF-deficient macrophages revealed reduced expression of the pattern recognition receptor dectin-1; restoration of dectin-1 expression recovered innate cytokine production and mycobacterial killing. Our data place MIF in a crucial upstream position in the innate immune response to mycobacteria and suggest that commonly occurring low expression MIF alleles confer an increased risk of TB disease in some populations.

Cryptococcus inositol utilization modulates the host protective immune response during brain infection.

BACKGROUND: Cryptococcus neoformans is the most common cause of fungal meningitis among individuals with HIV/AIDS, which is uniformly fatal without proper treatment. The underlying mechanism of disease development in the brain that leads to cryptococcal meningoencephalitis remains incompletely understood. We have previously demonstrated that inositol transporters (ITR) are required for Cryptococcus virulence. The itr1aΔ itr3cΔ double mutant of C. neoformans was attenuated for virulence in a murine model of intra-cerebral infection; demonstrating that Itr1a and Itr3c are required for full virulence during brain infection, despite a similar growth rate between the mutant and wild type strains in the infected brain. RESULTS: To understand the immune pathology associated with infection by the itr1aΔ itr3cΔ double mutant, we investigated the molecular correlates of host immune response during mouse brain infection. We used genome-wide transcriptome shotgun sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) methods to examine the host gene expression profile in the infected brain. Our results show that compared to the wild type, infection of mouse brains by the mutant leads to significant activation of cellular networks/pathways associated with host protective immunity. Most of the significantly differentially expressed genes (SDEG) are part of immune cell networks such as tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) regulon, indicating that infection by the mutant mounts a stronger host immune response compared to the wild type. Interestingly, a significant reduction in glucuronoxylomannan (GXM) secretion was observed in the itr1aΔ itr3cΔ mutant cells, indicating that inositol utilization pathways play a role in capsule production. CONCLUSIONS: Since capsule has been shown to impact the host response during Cryptococcus-host interactions, our results suggest that the reduced GXM production may contribute to the increased immune activation in the mutant-infected animals.

The causes and consequences of trained immunity in myeloid cells.

Conventionally, immunity in humans has been classified as innate and adaptive, with the concept that only the latter type has an immunological memory/recall response against specific antigens or pathogens. Recently, a new concept of trained immunity (a.k.a. innate memory response) has emerged. According to this concept, innate immune cells can exhibit enhanced responsiveness to subsequent challenges, after initial stimulation with antigen/pathogen. Thus, trained immunity enables the innate immune cells to respond robustly and non-specifically through exposure or re-exposure to antigens/infections or vaccines, providing enhanced resistance to unrelated pathogens or reduced infection severity. For example, individuals vaccinated with BCG to protect against tuberculosis were also protected from malaria and SARS-CoV-2 infections. Epigenetic modifications such as histone acetylation and metabolic reprogramming (e.g. shift towards glycolysis) and their inter-linked regulations are the key factors underpinning the immune activation of trained cells. The integrated metabolic and epigenetic rewiring generates sufficient metabolic intermediates, which is crucial to meet the energy demand required to produce proinflammatory and antimicrobial responses by the trained cells. These factors also determine the efficacy and durability of trained immunity. Importantly, the signaling pathways and regulatory molecules of trained immunity can be harnessed as potential targets for developing novel intervention strategies, such as better vaccines and immunotherapies against infectious (e.g., sepsis) and non-infectious (e.g., cancer) diseases. However, aberrant inflammation caused by inappropriate onset of trained immunity can lead to severe autoimmune pathological consequences, (e.g., systemic sclerosis and granulomatosis). In this review, we provide an overview of conventional innate and adaptive immunity and summarize various mechanistic factors associated with the onset and regulation of trained immunity, focusing on immunologic, metabolic, and epigenetic changes in myeloid cells. This review underscores the transformative potential of trained immunity in immunology, paving the way for developing novel therapeutic strategies for various infectious and non-infectious diseases that leverage innate immune memory.