Leishmania exploits host cAMP/EPAC/calcineurin signaling to induce an IL-33-mediated anti-inflammatory environment for the establishment of infection.

Host anti-inflammatory responses are critical for the progression of visceral leishmaniasis, and the pleiotropic cytokine interleukin (IL)-33 was found to be upregulated in infection. Here, we documented that IL-33 induction is a consequence of elevated cAMP-mediated exchange protein activated by cAMP (EPAC)/calcineurin-dependent signaling and essential for the sustenance of infection. Leishmania donovani-infected macrophages showed upregulation of IL-33 and its neutralization resulted in decreased parasite survival and increased inflammatory responses. Infection-induced cAMP was involved in IL-33 production and of its downstream effectors PKA and EPAC, only the latter was responsible for elevated IL-33 level. EPAC initiated Rap-dependent phospholipase C activation, which triggered the release of intracellular calcium followed by calcium/calmodulin complex formation. Screening of calmodulin-dependent enzymes affirmed involvement of the phosphatase calcineurin in cAMP/EPAC/calcium/calmodulin signaling-induced IL-33 production and parasite survival. Activated calcineurin ensured nuclear localization of the transcription factors, nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha required for IL-33 transcription, and we further confirmed this by chromatin immunoprecipitation assay. Administering specific inhibitors of nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha in BALB/c mouse model of visceral leishmaniasis decreased liver and spleen parasite burden along with reduction in IL-33 level. Splenocyte supernatants of inhibitor-treated infected mice further documented an increase in tumor necrosis factor alpha and IL-12 level with simultaneous decrease of IL-10, thereby indicating an overall disease-escalating effect of IL-33. Thus, this study demonstrates that cAMP/EPAC/calcineurin signaling is crucial for the activation of IL-33 and in effect creates anti-inflammatory responses, essential for infection.

Antileishmanial Activity of Cathelicidin and its Modulation by Leishmania donovani in a cAMP Response Element Modulator-Dependent Manner in Infection.

Concerns regarding toxicity and resistance of current drugs in visceral leishmaniasis have been reported. Antimicrobial peptides are considered to be promising candidates and among them human cathelicidin hCAP18/LL-37 showed significant parasite killing on drug-sensitive and resistant Leishmania promastigotes, in addition to its apoptosis-inducing role. Administration of hCAP18/LL-37 to infected macrophages also decreased parasite survival and increased the host favorable cytokine interleukin 12. However, 1,25-dihydroxyvitamin D3 (vitamin D3)-induced endogenous hCAP18/LL-37 production was hampered in infected THP-1 cells. Infection also suppressed the vitamin D3 receptor (VDR), transcription factor of hCAP18/LL-37. cAMP response element modulator (CREM), the repressor of VDR, was induced in infection, resulting in suppression of both VDR and cathelicidin expression. PGE2/cAMP/PKA axis was found to regulate CREM induction during infection and silencing CREM in infected cells and BALB/c mice led to decreased parasite survival. This study documents the antileishmanial potential of cathelicidin and further identifies CREM as a repressor of cathelicidin in Leishmania infection.

Translationally Controlled Tumor Protein-Mediated Stabilization of Host Antiapoptotic Protein MCL-1 Is Critical for Establishment of Infection by Intramacrophage Parasite Leishmania donovani.

In the early phase of infection, the intramacrophage pathogen Leishmania donovani protects its niche with the help of the antiapoptotic protein myeloid cell leukemia-1 (MCL-1). Whether Leishmania could exploit MCL-1, an extremely labile protein, at the late phase is still unclear. A steady translational level of MCL-1 observed up to 48 h postinfection and increased caspase-3 activity in MCL-1-silenced infected macrophages documented its importance in the late hours of infection. The transcript level of MCL-1 showed a sharp decline at 6 h postinfection, and persistent MCL-1 expression in cyclohexamide-treated cells negates the possibility of de novo protein synthesis, thereby suggesting infection-induced stability. Increased ubiquitination, a prerequisite for proteasomal degradation of MCL-1, was also found to be absent in the late hours of infection. Lack of interaction with its specific E3 ubiquitin ligase MULE (MCL-1 ubiquitin ligase E3) and specific deubiquitinase USP9X prompted us to search for blockade of the ubiquitin-binding site in MCL-1. To this end, TCTP (translationally controlled tumor protein), a well-known binding partner of MCL-1 and antiapoptotic regulator, was found to be strongly associated with MCL-1 during infection. Phosphorylation of TCTP, a requirement for MCL-1 binding, was also increased in infected macrophages. Knockdown of TCTP decreased MCL-1 expression and short hairpin RNA-mediated silencing of TCTP in an infected mouse model of visceral leishmaniasis showed decreased parasite burden and induction of liver cell apoptosis. Collectively, our investigation revealed a key mechanism of how L. donovani exploits TCTP to establish infection within the host.

Increasing Production Diversity and Diet Quality: Evidence from Bangladesh.

In the context of rural Bangladesh, we assess whether agriculture training alone, nutrition Behavior Communication Change (BCC) alone, combined agriculture training and nutrition BCC, or agriculture training and nutrition BCC combined with gender sensitization improve: (a) production diversity, either on household fields or through crop, livestock or aquaculture activities carried out near the family homestead and (b) diet diversity and the quality of household diets. All treatment arms were implemented by government employees. Implementation quality was high. No treatment increased production diversification of crops grown on fields. Treatment arms with agricultural training did increase the number of different crops grown in homestead gardens and the likelihood of any egg, dairy, or fish production but the magnitudes of these effect sizes were small. All agricultural treatment arms had, in percentage terms, large effects on measures of levels of homestead production. However, because baseline levels of production were low, the magnitude of these changes in absolute terms was modest. Nearly all treatment arms improved measures of food consumption and diet with the largest effects found when nutrition and agriculture training were combined. Relative to treatments combining agriculture and nutrition training, we find no significant impact of adding the gender sensitization on our measures of production diversity or diet quality. Interventions that combine agricultural training and nutrition BCC can improve both production diversity and diet quality, but they are not a panacea. They can, however, contribute towards better diets of rural households.

PD-1 negatively tunes macrophage immune activation by turning off JNK and STAT1 signaling: Exploited by Leishmania for its intra-macrophage survival.

The anti-inflammatory role of the programmed death-1 receptor (PD-1) is well appreciated. However, the mechanism of how PD-1 signaling inhibits the pro-inflammatory cytokine responses in macrophages, which is further exploited by Leishmania to foster their intracellular survival, was unknown. We found that among three major MAP kinases regulating immune activation, PD-1 signaling decreased only JNK phosphorylation without perturbing p38 and ERK. Inflammatory transcription factor STAT1 was also inhibited by PD-1. Association studies documented that SHP, the downstream phosphatase of PD-1, is directly responsible for the decreased phosphorylation of JNK and STAT1. JNK and STAT1 deactivation led to Elk-1/c-Fos inhibition, which significantly decreased IL-12 and TNF-α levels. Further investigation revealed c-Fos deactivation ultimately rendered transcription factor AP1 inactive and facilitating parasite-favorable anti-inflammatory environment.

Trust and distrust in low-income Michigan residents during the early COVID-19 pandemic: A qualitative study.

BACKGROUND: Trust and distrust have shaped health behaviour during the COVID-19 pandemic. Since the start of the pandemic, misinformation and polarization eroded trust across the United States. In states like Michigan, pandemic restrictions led to significant unrest. Michiganders also faced disproportionate morbidity and mortality from COVID-19 during this period. OBJECTIVE: The objective of this qualitative study was to understand the individual experiences of trust in low-income Michiganders during the early COVID-19 pandemic. PARTICIPANTS: Twenty-four participants at or below 200% of the federal poverty line who resided in Michigan were recruited for this study. APPROACH: Interviews were conducted during the winter of 2020 using a formal interview guide that addressed sources of information, perceptions of risk and exposure, protective behaviours and impacts of the pandemic at home, work and in receiving healthcare. RESULTS: Thematic analysis showed that themes of trust and distrust emerged in multiple facets of our participants' experiences, including in the context of information sources, the behaviours of others, health, financial security, employment and overall safety. Trust and distrust in low-income communities often stemmed from significant financial and economic vulnerabilities and instability in access to healthcare that was exacerbated in the pandemic. Furthermore, participant trust was shaped by internal (e.g., relationships with others) and external (e.g., source of information, social inequity) factors that influenced their perceptions and experiences during the pandemic. CONCLUSION: Trust has played an important role in many aspects of the experiences of low-income communities during the COVID-19 pandemic. This is important for clinicians to consider as COVID-19 becomes endemic, and trust continues to impact patients' approaches to vaccines, testing and treatment options. PATIENT OR PUBLIC CONTRIBUTION: This study was designed and conducted with the assistance and input of the members of the DECIDERS Steering Committee, a diverse statewide network of community members in Michigan. The DECIDERS team allows community members to have a voice in the design and conduct of health research, and collaborates with researchers to improve health across the state of Michigan.

Comparing delivery channels to promote nutrition-sensitive agriculture: A cluster-randomized controlled trial in Bangladesh.

We use a randomized controlled trial in rural Bangladesh to compare two models of delivering nutrition content jointly to husbands and wives: deploying female nutrition workers versus mostly male agriculture extension workers. Both approaches increased nutrition knowledge of men and women, household and individual diet quality, and women's empowerment. Intervention effects on agriculture and nutrition knowledge, agricultural production diversity, dietary diversity, women's empowerment, and gender parity do not significantly differ between models where nutrition workers versus agriculture extension workers provide the training. The exception is in an attitudes score, where results indicate same-sex agents may affect scores differently than opposite-sex agents. Our results suggest opposite-sex agents may not necessarily be less effective in providing training. In South Asia, where agricultural extension systems and the pipeline to those systems are male-dominated, training men to deliver nutrition messages may offer a temporary solution to the shortage of female extension workers and offer opportunities to scale and promote nutrition-sensitive agriculture. However, in both models, we find evidence that the presence of mothers-in-law within households modifies the programs' effectiveness on some nutrition, empowerment, and attitude measures, suggesting that accounting for other influential household members is a potential area for future programming.

Development and validation of a health and nutrition module for the project-level Women's Empowerment in Agriculture Index (pro-WEAI+HN).

Agricultural development projects increasingly aim to improve health and nutrition outcomes, often by engaging women. Although evidence shows such projects can improve women's and children's health and nutrition and empower women, little is known about their impacts on women's health- and nutrition-related agency and the extent to which impacts emerge through women's empowerment, largely due to a lack of instruments that measure the dimensions of women's agency that are directly relevant to health and nutrition outcomes. We developed an optional, complementary module for the project-level women's empowerment in agriculture index (pro-WEAI) to measure health- and nutrition-related agency (pro-WEAI + HN). Our method for developing related indicators used data collected from six agricultural development programmes implemented across Bangladesh, Burkina Faso and Mali (pooled sample = 12,114) and applied psychometric analysis (exploratory and confirmatory factor analysis) and the Alkire-Foster methodology. Results revealed seven indicators covering women's agency in the areas of her own health and diet; her health and diet during pregnancy; her child's diet; breastfeeding and weaning; purchasing food and health products; and acquiring food and health products. Multigroup confirmatory factor analysis revealed measurement invariance across contexts and samples. Tests of association (Cramer's V) and redundancy suggest that the pro-WEAI + HN indicators measured aspects of agency that are distinct from the core pro-WEAI. The uptake of these indicators in studies of nutrition-sensitive agricultural development projects may strengthen the evidence on how such programming can enhance women's empowerment to improve health and nutrition outcomes for themselves and their children.

Leishmania donovani Targets Host Transcription Factor NRF2 To Activate Antioxidant Enzyme HO-1 and Transcriptional Repressor ATF3 for Establishing Infection.

We showed previously that antioxidant enzyme heme oxygenase 1 (HO-1) is critical for Leishmania survival in visceral leishmaniasis. HO-1 inhibits host oxidative burst and inflammatory cytokine production, leading to parasite persistence. In the present study, screening of reported HO-1 transcription factors revealed that infection upregulated (4.1-fold compared to control [P < 0.001]) nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2). Silencing of NRF2 reduced both HO-1 expression and parasite survival. Investigation revealed that infection-induced transient reactive oxygen species (ROS) production dissociated NRF2 from its inhibitor KEAP1 and enabled phosphorylation-dependent nuclear translocation. Both NRF2 and HO-1 silencing in infection increased production of proinflammatory cytokines. But the level was greater in NRF2-silenced cells than in HO-1-silenced ones, suggesting the presence of other targets of NRF2. Another stress responsive transcription factor ATF3 is also induced (4.6-fold compared to control [P < 0.001]) by NRF2 during infection. Silencing of ATF3 reduced parasite survival (59.3% decrease compared to control [P < 0.001]) and increased proinflammatory cytokines. Infection-induced ATF3 recruited HDAC1 into the promoter sites of tumor necrosis factor alpha (TNF-α) and interleukin 12b (IL-12b) genes. Resulting deacetylated histones prevented NF-κB promoter binding, thereby reducing transcription of inflammatory cytokines. Administering the NRF2 inhibitor trigonelline hydrochloride to infected BALB/c mice resulted in reduced HO-1 and ATF3 expression, decreased spleen and liver parasite burdens, and increased proinflammatory cytokine levels. These results suggest that Leishmania upregulates NRF2 to activate both HO-1 and ATF3 for disease progression.

Crosstalk of PD-1 signaling with the SIRT1/FOXO-1 axis during the progression of visceral leishmaniasis.

Previously, we documented the role of the programmed death-1 (PD-1, also known as PDCD1) pathway in macrophage apoptosis and the downregulation of this signaling during infection by the intra-macrophage parasite Leishmania donovani However, we also found that, during the late phase of infection, PD-1 expression was significantly increased without activating host cell apoptosis; here we show that inhibition of PD-1 led to markedly decreased parasite survival, along with increased production of TNFα, IL-12, reactive oxygen species (ROS) and nitric oxide (NO). Increased PD-1 led to inactivation of AKT proteins resulting in nuclear sequestration of FOXO-1. Transfecting infected cells with constitutively active FOXO-1 (CA-FOXO) led to increased cell death, thereby suggesting that nuclear FOXO-1 might be inactivated. Infection significantly induced the expression of SIRT1, which inactivated FOXO-1 through deacetylation, and its knockdown led to increased apoptosis. SIRT1 knockdown also significantly decreased parasite survival along with increased production of TNFα, ROS and NO. Administration of the SIRT1 inhibitor sirtinol (10 mg/kg body weight) in infected mice decreased spleen parasite burden and a synergistic effect was found with PD-1 inhibitor. Collectively, our study shows that Leishmania utilizes the SIRT1/FOXO-1 axis for differentially regulating PD-1 signaling and, although they are interconnected, both pathways independently contribute to intracellular parasite survival.This article has an associated First Person interview with the first author of the paper.