A mouse model of Salmonella typhi infection.

Salmonella spp. are gram-negative flagellated bacteria that can cause food- and waterborne gastroenteritis and typhoid fever in humans. We now report that flagellin from Salmonella spp. is recognized in mouse intestine by Toll-like receptor 11 (TLR11). Absence of TLR11 renders mice more susceptible to infection by S. Typhimurium, with increased dissemination of the bacteria and enhanced lethality. Unlike S. Typhimurium, S. Typhi, a human obligatory pathogen that causes typhoid fever, is normally unable to infect mice. TLR11 is expressed in mice, but not in humans, and remarkably, we find that tlr11(-/-) mice are efficiently infected with orally administered S. Typhi. We also find that tlr11(-/-) mice can be immunized against S. Typhi. Therefore, tlr11(-/-) mice represent a small-animal model for the study of the immune response to S. Typhi and for the development of vaccines against this important human pathogen.

IL-17RA promotes pathologic epithelial inflammation in a mouse model of upper respiratory influenza infection.

The upper respiratory tract (nasopharynx or NP) is the first site of influenza replication, allowing the virus to disseminate to the lower respiratory tract or promoting community transmission. The host response in the NP regulates an intricate balance between viral control and tissue pathology. The hyper-inflammatory responses promote epithelial injury, allowing for increased viral dissemination and susceptibility to secondary bacterial infections. However, the pathologic contributors to influenza upper respiratory tissue pathology are incompletely understood. In this study, we investigated the role of interleukin IL-17 recetor A (IL-17RA) as a modulator of influenza host response and inflammation in the upper respiratory tract. We used a combined experimental approach involving IL-17RA-/- mice and an air-liquid interface (ALI) epithelial culture model to investigate the role of IL-17 response in epithelial inflammation, barrier function, and tissue pathology. Our data show that IL-17RA-/- mice exhibited significantly reduced neutrophilia, epithelial injury, and viral load. The reduced NP inflammation and epithelial injury in IL-17RA-/- mice correlated with increased resistance against co-infection by Streptococcus pneumoniae (Spn). IL-17A treatment, while potentiating the apoptosis of IAV-infected epithelial cells, caused bystander cell death and disrupted the barrier function in ALI epithelial model, supporting the in vivo findings.

Mitochondrial Oxidative Stress Regulates FOXP3+ T-Cell Activity and CD4-Mediated Inflammation in Older Adults with Frailty.

In healthy older adults, the immune system generally preserves its response and contributes to a long, healthy lifespan. However, rapid deterioration in immune regulation can lead to chronic inflammation, termed inflammaging, which accelerates pathological aging and diminishes the quality of life in older adults with frailty. A significant limitation in current aging research is the predominant focus on comparisons between young and older populations, often overlooking the differences between healthy older adults and those experiencing pathological aging. Our study elucidates the intricate immunological dynamics of the CD4/Treg axis in frail older adults compared to comparable age-matched healthy older adults. By utilizing publicly available RNA sequencing and single-cell RNA sequencing (scRNAseq) data from peripheral blood mononuclear cells (PBMCs), we identified a specific Treg cell subset and transcriptional landscape contributing to the dysregulation of CD4+ T-cell responses. We explored the molecular mechanisms underpinning Treg dysfunction, revealing that Tregs from frail older adults exhibit reduced mitochondrial protein levels, impairing mitochondrial oxidative phosphorylation. This impairment is driven by the TNF/NF-kappa B pathway, leading to cumulative inflammation. Further, we gained a deeper understanding of the CD4/Treg axis by predicting the effects of gene perturbations on cellular signaling networks. Collectively, these findings highlight the age-related relationship between mitochondrial dysfunction in the CD4/Treg axis and its role in accelerating aging and frailty in older adults. Targeting Treg dysfunction offers a critical basis for developing tailored therapeutic strategies aimed at improving the quality of life in older adults.

Proteomics Profiling Reveals Regulation of Immune Response to Salmonella enterica Serovar Typhimurium Infection in Mice.

Regulation of the immune response to Salmonella enterica serovar Typhimurium (S. Typhimurium) infection is a complex process, influenced by the interaction between genetic and environmental factors. Different inbred strains of mice exhibit distinct levels of resistance to S. Typhimurium infection, ranging from susceptible (e.g., C57BL/6J) to resistant (e.g., DBA/2J) strains. However, the underlying molecular mechanisms contributing to the host response remain elusive. In this study, we present a comprehensive proteomics profiling of spleen tissue from C57BL/6J and DBA/2J strains with different doses of S. Typhimurium infection by tandem mass tag labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (TMT-LC/LC-MS/MS). We identified and quantified 3,986 proteins, resulting in 475 differentially expressed proteins (DEPs) between C57BL/6J and DBA/2J strains. Functional enrichment analysis unveiled that the mechanisms of innate immune responses to S. Typhimurium infection could be associated with several signaling pathways, including the interferon (IFN) signaling pathway. We experimentally validated the roles of the IFN signaling pathway in the innate immune response to S. Typhimurium infection using an IFN-γ neutralization assay. We further illustrated the importance of macrophage and proinflammatory cytokines in the mechanisms underlying the resistance to S. Typhimurium using quantitative reverse transcription-PCR (qRT-PCR). Taken together, our results provided new insights into the genetic regulation of the immune response to S. Typhimurium infection in mice and might lead to the discovery of potential protein targets for controlling salmonellosis.

Cellular Heterogeneity and Molecular Reprogramming of the Host Response during Influenza Acute Lung Injury.

An exuberant host response contributes to influenza A virus (IAV) (or influenza)-mediated lung injury. However, despite significant information on the host response to IAV, the cellular framework and molecular interactions that dictate the development of acute injury in IAV-infected lungs remain incompletely understood. We performed an unbiased single-cell RNA sequencing (scRNAseq) analysis to examine the cellular heterogeneity and regulation of host responses in the IAV model of acute lung injury. At the cellular level, IAV infection promoted the overwhelming recruitment of monocytes that exhibited the cell differentiation trajectory to monocyte-derived macrophages. Together, monocytes and monocyte-derived myeloid cells constituted over 50% of the total immune cells in IAV-infected lungs. In contrast, IAV infection resulted in a significant loss of nonhematopoietic cells. Molecularly, our data show the multidimensional cell-cell communication dynamics of interferon and chemokine signaling between immune and nonimmune cells and the cell-specific molecular pathways regulating the host responses during IAV-induced lung injury. Our data provide a foundation for further exploring the mechanistic association of the IAV host response with acute lung injury. IMPORTANCE A dysregulated host response develops acute lung injury during IAV infection. However, the pathological immune mechanism(s) associated with acute lung injury during IAV infection is yet to be elucidated. In this study, we performed scRNAseq to examine the dynamics of host responses during the peak of IAV-mediated lung injury. At the cellular level, our data reveal significant myelopoiesis predominated by monocytes and macrophages and the simultaneous disruption of the nonhematopoietic cell framework, crucial for regulating inflammation and barrier integrity in IAV-infected lungs. Molecularly, we observed a complex cellular network involving cell-cell communications and a number of unique regulons dictating the outcome of interferon and chemokine responses during peak lung injury. Our data present a unique atlas of cellular changes and the regulation of global and cell-specific host responses during IAV infection. We expect that this information will open new avenues to identify targets for therapeutic intervention against IAV lung injury.

Recognition of profilin by Toll-like receptor 12 is critical for host resistance to Toxoplasma gondii.

Toll-like receptor 11 (TLR11) recognizes T. gondii profilin (TgPRF) and is required for interleukin-12 production and induction of immune responses that limit cyst burden in Toxoplasma gondii-infected mice. However, TLR11 only modestly affects survival of T. gondii-challenged mice. We report that TLR12, a previously uncharacterized TLR, also recognized TgPRF. TLR12 was sufficient for recognition of TgPRF by plasmacytoid dendritic cells (pDCs), whereas TLR11 and TLR12 were both required in macrophages and conventional DCs. In contrast to TLR11, TLR12-deficient mice succumb rapidly to T. gondii infection. TLR12-dependent induction of IL-12 and IFN-α in pDCs led to production of IFN-γ by NK cells. Consistent with this observation, the partial resistance of Tlr11(-/-) mice is lost upon pDC or NK cell depletion. Thus, TLR12 is critical for the innate immune response to T. gondii, and this TLR may promote host resistance by triggering pDC and NK cell function.

Microglial mTOR is Neuronal Protective and Antiepileptogenic in the Pilocarpine Model of Temporal Lobe Epilepsy.

Excessive activation of mammalian target of rapamycin (mTOR) signaling is epileptogenic in genetic epilepsy. However, the exact role of microglial mTOR in acquired epilepsy remains to be clarified. In the present study, we found that mTOR is strongly activated in microglia following excitatory injury elicited by status epilepticus. To determine the role of microglial mTOR signaling in excitatory injury and epileptogenesis, we generated mice with restrictive deletion of mTOR in microglia. Both male and female mice were used in the present study. We found that mTOR-deficient microglia lost their typical proliferative and inflammatory responses to excitatory injury, whereas the proliferation of astrocytes was preserved. In addition, mTOR-deficient microglia did not effectively engulf injured/dying neurons. More importantly, microglial mTOR-deficient mice displayed increased neuronal loss and developed more severe spontaneous seizures. These findings suggest that microglial mTOR plays a protective role in mitigating neuronal loss and attenuating epileptogenesis in the excitatory injury model of epilepsy.SIGNIFICANCE STATEMENT The mammalian target of rapamycin (mTOR) pathway is strongly implicated in epilepsy. However, the effect of mTOR inhibitors in preclinical models of acquired epilepsy is inconsistent. The broad presence of mTOR signaling in various brain cells could prevent mTOR inhibitors from achieving a net therapeutic effect. This conundrum has spurred further investigation of the cell type-specific effects of mTOR signaling in the CNS. We found that activation of microglial mTOR is antiepileptogenic. Thus, microglial mTOR activation represents a novel antiepileptogenic route that appears to parallel the proepileptogenic route of neuronal mTOR activation. This may explain why the net effect of mTOR inhibitors is paradoxical in the acquired models of epilepsy. Our findings could better guide the use of mTOR inhibitors in preventing acquired epilepsy.

T regulatory cells maintain intestinal homeostasis by suppressing γδ T cells.

Immune tolerance against enteric commensal bacteria is important for preventing intestinal inflammation. Deletion of phosphoinositide-dependent protein kinase 1 (Pdk1) in T cells via Cd4-Cre induced chronic inflammation of the intestine despite the importance of PDK1 in T cell activation. Analysis of colonic intraepithelial lymphocytes of PDK1-deficient mice revealed markedly increased CD8α(+) T cell receptor (TCR)γδ(+) T cells, including an interleukin-17 (IL-17)-expressing population. TCRγδ(+) T cells were responsible for the inflammatory colitis as shown by the fact that deletion of Tcrd abolished spontaneous colitis in the PDK1-deficient mice. This dysregulation of intestinal TCRγδ(+) T cells was attributable to a reduction in the number and functional capacity of PDK1-deficient T regulatory (Treg) cells. Adoptive transfer of wild-type Treg cells abrogated the spontaneous activation and proliferation of intestinal TCRγδ(+) T cells observed in PDK1-deficient mice and prevented the development of colitis. Therefore, suppression of intestinal TCRγδ(+) T cells by Treg cells maintains enteric immune tolerance.

Attenuation of in vitro host-pathogen interactions in quinolone-resistant Salmonella Typhi mutants.

OBJECTIVES: The relationship between quinolone resistance acquisition and invasion impairment has been studied in some Salmonella enterica serovars. However, little information has been reported regarding the invasive human-restricted pathogen Salmonella Typhi. The aim of this study was to investigate the molecular mechanisms of quinolone resistance acquisition and its impact on virulence in this serovar. METHODS: Two antibiotic-resistant mutants (Ty_c1 and Ty_c2) were generated from a Salmonella Typhi clinical isolate (Ty_wt). The three strains were compared in terms of antimicrobial susceptibility, molecular mechanisms of resistance, gene expression of virulence-related factors, ability to invade eukaryotic cells (human epithelial cells and macrophages) and cytokine production. RESULTS: Multidrug resistance in Ty_c2 was attributed to AcrAB/TolC overproduction, decreased OmpF (both mediated by the mar regulon) and decreased OmpC. The two mutants showed a gradually reduced expression of virulence-related genes (invA, hilA, hilD, fliC and fimA), correlating with decreased motility, reduced infection of HeLa cells and impaired uptake by and intracellular survival in human macrophages. Moreover, Ty_c2 also showed reduced tviA expression. Additionally, we revealed a significant reduction in TNF-α and IL-1ß production and decreased NF-κB activation. CONCLUSIONS: In this study, we provide an in-depth characterization of the molecular mechanisms of antibiotic resistance in the Salmonella Typhi serovar and evidence that acquisition of antimicrobial resistance is concomitantly detected with a loss of virulence (epithelial cell invasion, macrophage phagocytosis and cytokine production). We suggest that the low prevalence of clinical isolates of Salmonella Typhi highly resistant to ciprofloxacin is due to poor immunogenicity and impaired dissemination ability of these isolates.

Immunological Signatures for Early Detection of Human Head and Neck Squamous Cell Carcinoma through RNA Transcriptome Analysis of Blood Platelets.

Although there has been a reduction in head and neck squamous cell carcinoma occurrence, it continues to be a serious global health concern. The lack of precise early diagnostic biomarkers and postponed diagnosis in the later stages are notable constraints that contribute to poor survival rates and emphasize the need for innovative diagnostic methods. In this study, we employed machine learning alongside weighted gene co-expression network analysis (WGCNA) and network biology to investigate the gene expression patterns of blood platelets, identifying transcriptomic markers for HNSCC diagnosis. Our comprehensive examination of publicly available gene expression datasets revealed nine genes with significantly elevated expression in samples from individuals diagnosed with HNSCC. These potential diagnostic markers were further assessed using TCGA and GTEx datasets, demonstrating high accuracy in distinguishing between HNSCC and non-cancerous samples. The findings indicate that these gene signatures could revolutionize early HNSCC identification. Additionally, the study highlights the significance of tumor-educated platelets (TEPs), which carry RNA signatures indicative of tumor-derived material, offering a non-invasive source for early-detection biomarkers. Despite using platelet and tumor samples from different individuals, our results suggest that TEPs reflect the transcriptomic and epigenetic landscape of tumors. Future research should aim to directly correlate tumor and platelet samples from the same patients to further elucidate this relationship. This study underscores the potential of these biomarkers in transforming early diagnosis and personalized treatment strategies for HNSCC, advocating for further research to validate their predictive and therapeutic potential.

The chemokine receptor CXCR3 promotes CD8+ T cell-dependent lung pathology during influenza pathogenesis.

The dual role of CD8+ T cells in influenza control and lung pathology is increasingly appreciated. To explore whether protective and pathological functions can be linked to specific subsets, we dissected CD8+ T responses in influenza-infected murine lungs. Our single-cell RNA-sequencing (scRNA-seq) analysis revealed notable diversity in CD8+ T subpopulations during peak viral load and infection-resolved state. While enrichment of a Cxcr3hi CD8+ T effector subset was associated with a more robust cytotoxic response, both CD8+ T effector and central memory exhibited equally potent effector potential. The scRNA-seq analysis identified unique regulons regulating the cytotoxic response in CD8+ T cells. The late-stage CD8+ T blockade in influenza-cleared lungs or continuous CXCR3 blockade mitigated lung injury without affecting viral clearance. Furthermore, adoptive transfer of wild-type CD8+ T cells exacerbated influenza lung pathology in Cxcr3-/- mice. Collectively, our data imply that CXCR3 interception could have a therapeutic effect in preventing influenza-linked lung injury.

Age-related dysregulation of intestinal epithelium fucosylation is linked to an increased risk of colon cancer.

Colon cancer affects people of all ages. However, its frequency, as well as the related morbidity and mortality, are high among older adults. The complex physiological changes in the aging gut substantially limit the development of cancer therapies. Here, we identify a potentially unique intestinal microenvironment that is linked with an increased risk of colon cancer in older adults. Our findings show that aging markedly influenced persistent fucosylation of the apical surfaces of intestinal epithelial cells, which resulted in a favorable environment for tumor growth. Furthermore, our findings shed light on the importance of the host-commensal interaction, which facilitates the dysregulation of fucosylation and promotes tumor growth as people get older. We analyzed colonic microbial populations at the species level to find changes associated with aging that could contribute to the development of colon cancer. Analysis of single-cell RNA-sequencing data from previous publications identified distinct epithelial cell subtypes involved in dysregulated fucosylation in older adults. Overall, our study provides compelling evidence that excessive fucosylation is associated with the development of colon cancer, that age-related changes increase vulnerability to colon cancer, and that a dysbiosis in microbial diversity and metabolic changes in the homeostasis of older mice dysregulate fucosylation levels with age.

Performance enhancement of solar photovoltaic (PV) module using a novel flat plate (NFP) glass cover by reducing the effect of bird dropping (BD) settlement.

A massive bird dropping (BD) deposition on the common rectangular flat plate (RFP) of photovoltaic (PV) module is a matter of great concern in Western Rajasthan (WR) that diminish the overall energy production capacity of the system remarkably. In this research article, a prototype novel flat plate (NFP) design of a front glass cover of PV module is proposed to prevent the impact of BD settlement by the restriction of bird's sitting/movement on the front glass cover. In this regard, the performance analysis of PV module with common RFP and newly designed NFP glass covers has been assessed at the different inclination ß° (0-90). The BD accumulation onto the both glass covers was explored by the optical transmittance profiles at the different tilt angles, i.e., explained by bird movement on each flat glass surfaces. Consequently, a significant amount of output electric energy has been gained in NFP design rather than RFP corresponding to particular tilt regions TR I (0° ≤ ß ≤ 25°), TR II (25° ≤ ß ≤ 60°), and TR III (60° ≤ ß ≤ 90°). According to the results achieved, an excellent level of improvement in average power loss, ~ 97.85%, corresponding to optimal TR (III) has been detected by employing NFP glass collector.

Promoting autophagy to mitigate coronavirus disease pathology in the elderly.

In this commentary, we highlight autophagy's important function, while identifying potential therapeutic targets for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the elderly. Autophagy's decline in the elderly causes increased cell senescence and a dysregulated immune system. As this demographic often faces decreased vaccine-provided immunity, coronavirus disease 2019 treatments must be developed. We discuss a recent study by Acharya et al. (2022) that found that SF2523 induced autophagy, reducing SARS-CoV-2 replication. Furthermore, across varying dosages, SF2523 was shown to have a synergistic effect with remdesivir or MU-UNMC. Consequently, we believe that SF2523, alone or with other anti-virals, is a promising potential therapeutic for preventing SARS-CoV-2-related mortalities.

Interferon-γ promotes monocyte-mediated lung injury during influenza infection.

Influenza A virus (IAV) infection triggers an exuberant host response that promotes acute lung injury. However, the host response factors that promote the development of a pathologic inflammatory response to IAV remain incompletely understood. In this study, we identify an interferon-γ (IFN-γ)-regulated subset of monocytes, CCR2+ monocytes, as a driver of lung damage during IAV infection. IFN-γ regulates the recruitment and inflammatory phenotype of CCR2+ monocytes, and mice deficient in CCR2 (CCR2-/-) or IFN-γ (IFN-γ-/-) exhibit reduced lung inflammation, pathology, and disease severity. Adoptive transfer of wild-type (WT) (IFN-γR1+/+) but not IFN-γR1-/- CCR2+ monocytes restore the WT-like pathological phenotype of lung damage in IAV-infected CCR2-/- mice. CD8+ T cells are the main source of IFN-γ in IAV-infected lungs. Collectively, our data highlight the requirement of IFN-γ signaling in the regulation of CCR2+ monocyte-mediated lung pathology during IAV infection.

An Account of Immune Senescence in the Clinical Pathophysiology of COVID-19 Infection in Aging.

Worldwide COVID-19 infection poses an enormous risk to public health and an alarming global socioeconomic burden. The impact of the COVID-19 pandemic on individuals with underlying health conditions as well as on the elderly population is extensive and effective strategies are needed to understand the mechanism behind it. Cellular senescence defines as an irreversible cell cycle arrest due to DNA damage leading to accumulation of senescent cells in the elderly population and may result in worsening of COVID-19 mediated increased mortality. However, whether this variation in senescence levels, in different aged populations, translation to COVID-19 infection is unknown. The spike protein of SARS-CoV-2 has been recently identified to be responsible for inducing pathogenic signals, although a clear understanding of how the host receptor interacts with SARS-CoV-2 protein and mediates the immune responses is not clear. In this review, we address the epidemiology of SARS-CoV-2 and the cellular senescence responding immune response to pathogenic SARS-CoV-2. We provide a prospective summary of what to expect and how to brace the possible immunological strategy to protect against COVID-19 infection. The review majorly explores an underline mechanism of how senescent cells trigger a hyperimmune inflammatory response and cause high mortality in aging people could serve as a potential aid to alleviate the treatment for elderly battling COVID-19 infection.

Deficiency of Microglial Autophagy Increases the Density of Oligodendrocytes and Susceptibility to Severe Forms of Seizures.

Excessive activation of mTOR in microglia impairs CNS homeostasis and causes severe epilepsy. Autophagy constitutes an important part of mTOR signaling. The contribution of microglial autophagy to CNS homeostasis and epilepsy remains to be determined. Here, we report that ATG7KO mice deficient for autophagy in microglia display a marked increase of myelination markers, a higher density of mature oligodendrocytes (ODCs), and altered lengths of the nodes of Ranvier. Moreover, we found that deficiency of microglial autophagy (ATG7KO) leads to increased seizure susceptibility in three seizure models (pilocarpine, kainic acid, and amygdala kindling). We demonstrated that ATG7KO mice develop severe generalized seizures and display nearly 100% mortality to convulsions induced by pilocarpine and kainic acid. In the amygdala kindling model, we observed significant facilitation of contralateral propagation of seizures, a process underlying the development of generalized seizures. Taken together, our results reveal impaired microglial autophagy as a novel mechanism underlying altered homeostasis of ODCs and increased susceptibility to severe and fatal generalized seizures.

CD40 signaling is impaired in L. major-infected macrophages and is rescued by a p38MAPK activator establishing a host-protective memory T cell response.

Leishmania, a protozoan parasite, lives and multiplies as amastigote within macrophages. It is proposed that the macrophage expressed CD40 interacts with CD40 ligand on T cells to induce IFN-gamma, a Th1-type cytokine that restricts the amastigote growth. Here, we demonstrate that CD40 cross-linking early after infection resulted in inducible nitric oxide synthetase type-2 (iNOS2) induction and iNOS2-dependent amastigote elimination. Although CD40 expression remained unaltered on L. major-infected macrophages, delay in the treatment of macrophages or of mice with anti-CD40 antibody resulted in significant reduction in iNOS2 expression and leishmanicidal function suggesting impaired CD40 signaling in Leishmania infection. The inhibition of CD40-induced iNOS2 expression by SB203580, a p38-mitogen activated protein kinase (p38MAPK)-specific inhibitor, and the reversal of the inhibition by anisomycin, a p38MAPK activator, suggested a crucial role of p38MAPK in CD40 signaling. Indeed, the CD40-induced p38MAPK phosphorylation, iNOS2 expression and anti-leishmanial function were impaired in Leishmania-infected macrophages but were restored by anisomycin. Anisomycin's effects were reversed by SB203580 emphasizing the role of p38MAPK in CD40-induced iNOS2-dependent leishmanicidal function. Anisomycin administration in L. major-infected BALB/c mice resulted in significant reduction in the parasite load and established a host-protective Th1-type memory response. Also implicated in these findings is a scientific rationale to define novel anti-parasite drug targets and to bypass the problem of drug resistance.

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn's-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn's-associated intestinal fibrosis.