Conventional Tregs in treatment-naïve rheumatoid arthritis are deficient in suppressive function with an increase in percentage of CXCR3 and CCR6 expressing Tregs.

In rheumatoid arthritis (RA), immune homeostasis is maintained by T regulatory cells (Tregs) that in an inflammatory milieu can change towards T-helper-like phenotypes (Th-like Tregs). Our aim was to examine the phenotypic and functional characteristics of CD4+CD25+CD127lo/- Tregs, Th-like Tregs and T effector (Teff) cells in the peripheral blood (PB) and synovial fluid (SF) of treatment-naïve early RA, as compared to osteoarthritis (OA) and healthy control (HC) peripheral blood. Frequencies of Tregs, CXCR3, CCR6 expressing Tregs (Th-like Tregs), and Teff cells were analyzed using flow cytometry in RA (n = 80), OA (n = 20), and HC (n = 40). Cytokine concentrations of the respective T cell subsets in plasma and SF were measured using flow cytometric bead array. Tregs sorted from RA and HC PB using magnetic beads were analyzed for functional capacities by CFSE proliferation assay and FOXP3 gene expression using real-time PCR. We observed that the frequencies of Th17 cells in PB and SF were significantly higher in RA when compared to HC, whereas Tregs were lower in PB and high in SF compared to HC and OA respectively. Th1- and Th17-related pro-inflammatory cytokines IL12p70, INF-γ, TNF-α, and IL-6, and IL-17A were significantly higher in the plasma and SF of RA. Tregs expressing CXCR3 (Th1-like Tregs) and CCR6 (Th17-like Treg) were significantly higher in PB and SF of RA compared to controls and was positively associated with seropositivity and disease activity. Treg cells isolated from peripheral blood of RA showed decreased function and reduced FOXP3 gene expression compared to HC. In our study, we have demonstrated higher frequencies of Th1 and Th17 cells and increased circulatory and SF pro-inflammatory cytokines (IL12P70, INF-γ, IL-6, IL-17A, and TNF-α) in RA. This inflammatory milieu might alter total Tregs frequencies and influence conversion of Tregs into Th-like Tregs.

Not business as usual: Engaging the corporate sector in India's TB elimination efforts.

India has the highest global burden of tuberculosis (TB), accounting for a quarter of the worldwide TB disease incidence. Given the magnitude of India's epidemic, TB has enormous economic implications. Indeed, the majority of individuals with TB disease are in their prime years of economic productivity. Absenteeism and employee turnover due to TB have economic ramifications for employers. Furthermore, TB can easily spread in the workplace and compound the economic impact. Employers who fund workplace, community, or national TB initiatives stand to gain directly and also enjoy reputational benefits, which are important in the era of socially conscious investing. Corporate social responsibility laws in India and tax incentives can be leveraged to bring the logistical networks, reach, and innovative spirit of the private sector to bear on India's formidable TB epidemic. In this perspective piece, we explore the economic impacts of TB; opportunities for and benefits from businesses contributing to TB elimination efforts; and strategies to enlist India's corporate sector in the fight against TB.

Comparison of IGRA and TST in the diagnosis of latent tuberculosis among women of reproductive age in South India.

BACKGROUND: Latent tuberculosis infection (LTBI) is a mycobacterial infection defined on the basis of cellular immune response to mycobacterial antigens. The tuberculin skin test (TST) and the Interferon-Gamma Release Assay (IGRA) are the two tests currently used to establish the diagnosis of LTB. Literature suggests that a study regarding tuberculosis (TB) infection among women of reproductive age group is limited. METHODS: Female household contact, married, aged 18-49 years underwent written consent form and are screened for LTBI using the TST and IGRA. Participants are injected with TST [5 tuberculin unit (TU), purified protein derivative (PPD)] and IGRA [QuantiFERON®-TB Gold Plus kit (QFT-Plus)]. All the household contacts were followed-up for one year for incident TB cases. Statistical analysis was done using STATA version 14 (StataCorp., Texas, USA). Cohen's kappa test was used to determine the agreement between two tests. RESULTS: The prevalence of LTBI was found to be 69% (either TST or IGRA positive). Positivity rate of IGRA was higher when compared to that of TST. Out of 139 participants, 68 (49%) tested positive for TST, 80 (57.6%) tested positive for IGRA and 52 (37.4%) tested positive for both. Discordant results were observed in about two fifth of the study population and there was poor agreement between the two tests. CONCLUSION: Longitudinal studies are required to detect incident TB cases to evaluate the usefulness of these tests. The study was found that IGRA is more consistent to diagnosis of latent tuberculosis infection than the TST. Such studies can also be performed in varied settings among different populations which would help us to improve the diagnosis of LTBI and consequently help in TB control.

Corrigendum: Malnutrition leads to increased inflammation and expression of tuberculosis risk signatures in recently exposed household contacts of pulmonary tuberculosis.

[This corrects the article DOI: 10.3389/fimmu.2022.1011166.].

Malnutrition leads to increased inflammation and expression of tuberculosis risk signatures in recently exposed household contacts of pulmonary tuberculosis.

Background: Most individuals exposed to Mycobacterium tuberculosis (Mtb) develop latent tuberculosis infection (LTBI) and remain at risk for progressing to active tuberculosis disease (TB). Malnutrition is an important risk factor driving progression from LTBI to TB. However, the performance of blood-based TB risk signatures in malnourished individuals with LTBI remains unexplored. The aim of this study was to determine if malnourished and control individuals had differences in gene expression, immune pathways and TB risk signatures. Methods: We utilized data from 50 tuberculin skin test positive household contacts of persons with TB - 18 malnourished participants (body mass index [BMI] < 18.5 kg/m2) and 32 controls (individuals with BMI ≥ 18.5 kg/m2). Whole blood RNA-sequencing was conducted to identify differentially expressed genes (DEGs). Ingenuity Pathway Analysis was applied to the DEGs to identify top canonical pathways and gene regulators. Gene enrichment methods were then employed to score the performance of published gene signatures associated with progression from LTBI to TB. Results: Malnourished individuals had increased activation of inflammatory pathways, including pathways involved in neutrophil activation, T-cell activation and proinflammatory IL-1 and IL-6 cytokine signaling. Consistent with known association of inflammatory pathway activation with progression to TB disease, we found significantly increased expression of the RISK4 (area under the curve [AUC] = 0.734) and PREDICT29 (AUC = 0.736) progression signatures in malnourished individuals. Conclusion: Malnourished individuals display a peripheral immune response profile reflective of increased inflammation and a concomitant increased expression of risk signatures predicting progression to TB. With validation in prospective clinical cohorts, TB risk biomarkers have the potential to identify malnourished LTBI for targeted therapy.

Brugia malayi Microfilariae Induce Autophagy through an Interferon-γ Dependent Mechanism on Human Monocytes.

Monocyte dysfunction in helminth infection is one of the mechanisms proposed to explain the diminished parasite antigen-specific T cell responses seen with patent filarial infection. In fact, monocytes from filariae-infected individuals demonstrate internalized filarial antigens and, as a consequence, express inhibitory surface molecules and have diminished cytokine production. To investigate the mechanisms underlying monocyte dysfunction in filarial infections, purified human monocytes were exposed to live microfilariae (mf) of Brugia malayi, and the mRNA and protein expression of important inhibitory and/or autophagy-related molecules were assessed. Our data indicate that mf-induced autophagy in human monocytes shown by the formation of autophagic vesicles, by the upregulation in the mRNA expression of autophagy-related genes BCN1, LC3B, ATG5, ATG7 (P < 0.05), and by increase in the levels of LC3B protein. Furthermore, this mf-induced autophagy increased the levels of monocyte CD206 expression. In addition, mf significantly induced the frequency of interferon (IFN)-γ+ human monocytes and at the same time induced the mRNA expression of indoleamine 2,3-dioxygenase (IDO) through an IFN-γ-dependent mechanism; significantly enhanced tryptophan degradation (an indicator of IDO activity; P < 0.005). Interestingly, this autophagy induction by mf in monocytes was IFN-γ-dependent but IDO-independent as was reversed by anti-IFN-γ but not by an IDO inhibitor. Our data collectively suggest that mf of Brugia malayi regulate the function of monocytes by induction of IDO and IFN-γ, induce autophagy through an IFN-γ-dependent mechanism, and increase M2 phenotype through induction of autophagy; all acting in concert to drive monocyte dysfunction.

Nutritional Supplementation Would Be Cost-Effective for Reducing Tuberculosis Incidence and Mortality in India: The Ration Optimization to Impede Tuberculosis (ROTI-TB) Model.

BACKGROUND: Undernutrition is the leading cause of tuberculosis (TB) in India and is associated with increased TB mortality. Undernutrition also decreases quality of life and economic productivity. METHODS: We assessed the cost-effectiveness of providing augmented rations to undernourished Indians through the government's Targeted Public Distribution System (TPDS). We used Markov state transition models to simulate disease progression and mortality among undernourished individuals in 3 groups: general population, household contacts (HHCs) of people living with TB, and persons living with human immunodeficiency virus (HIV). The models calculate costs and outcomes (TB cases, TB deaths, and disability-adjusted life years [DALYs]) associated with a 2600 kcal/day diet for adults with body mass index (BMI) of 16-18.4 kg/m2 until they attain a BMI of 20 kg/m2 compared to a status quo scenario wherein TPDS rations are unchanged. We employed deterministic and probabilistic sensitivity analyses to test result robustness. RESULTS: Over 5 years, augmented rations could avert 81% of TB cases and 88% of TB deaths among currently undernourished Indians. Correspondingly, this intervention could forestall 78% and 48% of TB cases and prevent 88% and 70% of deaths among undernourished HHCs and persons with HIV, respectively. Augmented rations resulted in 10-fold higher resolution of undernutrition and were highly cost-effective with (incremental cost-effectiveness ratio [ICER] of $470/DALY averted). ICER was lower for HHCs ($360/DALY averted) and the HIV population ($250/DALY averted). CONCLUSIONS: A robust nutritional intervention would be highly cost-effective in reducing TB incidence and mortality while reducing chronic undernutrition in India.

Tuberculosis-Learning the Impact of Nutrition (TB LION): protocol for an interventional study to decrease TB risk in household contacts.

BACKGROUND: Comorbidities such as undernutrition and parasitic infections are widespread in India and other tuberculosis (TB)-endemic countries. This study examines how these conditions as well as food supplementation and parasite treatment might alter immune responses to Mycobacterium tuberculosis (Mtb) infection and risk of progression to TB disease. METHODS: This is a 5-year prospective clinical trial at Jawaharlal Institute of Post Graduate Medical Education and Research in Puducherry, Tamil Nadu, India. We aim to enroll 760 household contacts (HHC) of adults with active TB in order to identify 120 who are followed prospectively for 2 years: Thirty QuantiFERON-TB Gold Plus (QFT-Plus) positive HHCs ≥ 18 years of age in four proposed groups: (1) undernourished (body mass index [BMI] < 18.5 kg/m2); (2) participants with a BMI ≥ 18.5 kg/m2 who have a parasitic infection (3) undernourished participants with a parasitic infection and (4) controls-participants with BMI ≥ 18.5 kg/m2 and without parasitic infection. We assess immune response at baseline and after food supplementation (for participants with BMI < 18.5 kg/m2) and parasite treatment (for participants with parasites). Detailed nutritional assessments, anthropometry, and parasite testing through polymerase chain reaction (PCR) and microscopy are performed. In addition, at serial time points, these samples will be further analyzed using flow cytometry and whole blood transcriptomics to elucidate the immune mechanisms involved in disease progression. CONCLUSIONS: This study will help determine whether undernutrition and parasite infection are associated with gene signatures that predict risk of TB and whether providing nutritional supplementation and/or treating parasitic infections improves immune response towards this infection. This study transcends individual level care and presents the opportunity to benefit the population at large by analyzing factors that affect disease progression potentially reducing the overall burden of people who progress to TB disease. Trial registration ClinicalTrials.gov; NCT03598842; Registered on July 26, 2018; https://clinicaltrials.gov/ct2/show/NCT03598842.

Patrolling Monocytes Control NK Cell Expression of Activating and Stimulatory Receptors to Curtail Lung Metastases.

The role of nonclassical, patrolling monocytes in lung tumor metastasis and their functional relationships with other immune cells remain poorly defined. Contributing to these gaps in knowledge is a lack of cellular specificity in commonly used approaches for depleting nonclassical monocytes. To circumvent these limitations and study the role of patrolling monocytes in melanoma metastasis to lungs, we generated C57BL/6J mice in which the Nr4a1 superenhancer E2 subdomain is ablated (E2 -/- mice). E2 -/- mice lack nonclassical patrolling monocytes but preserve classical monocyte and macrophage numbers and functions. Interestingly, NK cell recruitment and activation were impaired, and metastatic burden was increased in E2 -/-mice. E2 -/- mice displayed unchanged "educated" (CD11b+CD27+) and "terminally differentiated" (CD11b+CD27-) NK cell frequencies. These perturbations were accompanied by reduced expression of stimulatory receptor Ly49D on educated NK cells and increased expression of inhibitory receptor NKG2A/CD94 on terminally differentiated NK cells. Thus, our work demonstrates that patrolling monocytes play a critical role in preventing lung tumor metastasis via NK cell recruitment and activation.

Nonclassical Monocytes in Health and Disease.

Monocytes are innate blood cells that maintain vascular homeostasis and are early responders to pathogens in acute infections. There are three well-characterized classes of monocytes: classical (CD14+CD16- in humans and Ly6Chi in mice), intermediate (CD14+CD16+ in humans and Ly6C+Treml4+ in mice), and nonclassical (CD14-CD16+ in humans and Ly6Clo in mice). Classical monocytes are critical for the initial inflammatory response. Classical monocytes can differentiate into macrophages in tissue and can contribute to chronic disease. Nonclassical monocytes have been widely viewed as anti-inflammatory, as they maintain vascular homeostasis. They are a first line of defense in recognition and clearance of pathogens. However, their roles in chronic disease are less clear. They have been shown to be protective as well as positively associated with disease burden. This review focuses on the state of the monocyte biology field and the functions of monocytes, particularly nonclassical monocytes, in health and disease.

Similarities and differences between helminth parasites and cancer cell lines in shaping human monocytes: Insights into parallel mechanisms of immune evasion.

A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma) or to live microfilariae (mf) of Brugia malayi-a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1ß), M2-associated (CCL13, CD206), Mreg-associated (IL-10, TGF-ß), and angiogenesis associated (MMP9, VEGF) genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1ß, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.

Microfilariae of Brugia malayi Inhibit the mTOR Pathway and Induce Autophagy in Human Dendritic Cells.

Immune modulation is a hallmark of patent filarial infection, including suppression of antigen-presenting cell function and downmodulation of filarial antigen-specific T cell responses. The mammalian target of rapamycin (mTOR) signaling pathway has been implicated in immune regulation, not only by suppressing T cell responses but also by regulating autophagy (through mTOR sensing amino acid availability). Global proteomic analysis (liquid chromatography-tandem mass spectrometry) of microfilaria (mf)-exposed monocyte-derived dendritic cells (DC) indicated that multiple components of the mTOR signaling pathway, including mTOR, eIF4A, and eIF4E, are downregulated by mf, suggesting that mf target this pathway for immune modulation in DC. Utilizing Western blot analysis, we demonstrate that similar to rapamycin (a known mTOR inhibitor), mf downregulate the phosphorylation of mTOR and its regulatory proteins, p70S6K1 and 4E-BP1, a process essential for DC protein synthesis. As active mTOR signaling regulates autophagy, we examined whether mf exposure alters autophagy-associated processes. mf-induced autophagy was reflected in marked upregulation of phosphorylated Beclin 1, known to play an important role in both autophagosome formation and autolysosome fusion, in induction of LC3II, a marker of autophagosome formation, and in induced degradation of p62, a ubiquitin-binding protein that aggregates protein in autophagosomes and is degraded upon autophagy that was reduced significantly by mf exposure and by rapamycin. Together, these results suggest that Brugia malayi mf employ mechanisms of metabolic modulation in DC to influence the regulation of the host immune response by downregulating mTOR signaling, resulting in increased autophagy. Whether this is a result of the parasite-secreted rapamycin homolog is currently under study.

Incomplete deletion of IL-4Rα by LysM(Cre) reveals distinct subsets of M2 macrophages controlling inflammation and fibrosis in chronic schistosomiasis.

Mice expressing a Cre recombinase from the lysozyme M-encoding locus (Lyz2) have been widely used to dissect gene function in macrophages and neutrophils. Here, we show that while naïve resident tissue macrophages from IL-4Rαf(lox/delta)LysM(Cre) mice almost completely lose IL-4Rα function, a large fraction of macrophages elicited by sterile inflammatory stimuli, Schistosoma mansoni eggs, or S. mansoni infection, fail to excise Il4rα. These F4/80(hi)CD11b(hi) macrophages, in contrast to resident tissue macrophages, express lower levels of Lyz2 explaining why this population resists LysM(Cre)-mediated deletion. We show that in response to IL-4 and IL-13, Lyz2(lo)IL-4Rα(+) macrophages differentiate into an arginase 1-expressing alternatively-activated macrophage (AAM) population, which slows the development of lethal fibrosis in schistosomiasis. In contrast, we identified Lyz2(hi)IL-4Rα(+) macrophages as the key subset of AAMs mediating the downmodulation of granulomatous inflammation in chronic schistosomiasis. Our observations reveal a limitation on using a LysMCre mouse model to study gene function in inflammatory settings, but we utilize this limitation as a means to demonstrate that distinct populations of alternatively activated macrophages control inflammation and fibrosis in chronic schistosomiasis.