bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR4 abrogates Th1 immune response against L. infantum infection through IRF1 and IFN-β-dependent mechanism preventing immunopathology
Authors: Sacramento Laís Amorim , Benevides Luciana , Maruyama Sandra , Tavares Lucas , Fukutani Kiyoshi , Cunha Fernando , Almeida Roque , Silva João , Carregaro Vanessa
Published: January 2023

Toll-like receptors (TLRs) are thought to be among the most ancient pathogen recognition systems, and these receptors directly interact with Leishmania species during the initial sensing of these parasites and mount the subsequent adaptive immune response. Herein, we aimed to evaluate the role of TLR4 during visceral leishmaniasis (VL) induced by Leishmania infantum. Initially, we observed that tlr4 messenger RNA (mRNA) expression was detected only the later stage of infection in target organs of VL, suggesting that TLR4 contributes significantly during the chronic phases of the disease. We have shown that Tlr4-deficient mice harbor fewer parasites in the spleen and liver than wild-type mice. TLR4 deficiency enhanced the Th1 immune response against the parasite, correlating with the increased activation of dendritic cells (DCs). Transcription analysis demonstrated that IRF1 and IFN-β are downstream of TLR4 after infection. Accordingly, IRF1- and IFNAR-genetically deficient mice harbored fewer parasites in the target organs than wild-type mice due to the increased Th1 response. The absence of TLR4 or IFNAR increases the serum transaminase level of infected mice. In addition, IFN-β acts directly on Th1 cells to limit IFN-γ production, without affecting the cell proliferation. Importantly, TLR4 and type I IFN (IFN-I) were positively modulated in asymptomatic subjects compared to VL patients. Thus, the TLR4-IRF1 pathway culminates in IFN-β production as a mechanism for dampening the chronic inflammatory process and preventing immunopathology development.
Neutrophils transiently harbor Mycobacterium tuberculosis and represent a metabolically permissive niche for bacterial growth
Authors: Huang Lu , Russell David
Published: January 2023

Neutrophils are rapidly recruited to sites of mycobacterial infection, where they phagocytose bacilli. At the early stage of infection, Mycobacterium tuberculosis is predominantly present in neutrophils, which are also frequently infected in human TB. However, neutrophils are less well studied than other components of the host response to Mtb and much of the available data are conflicting. To better understand the role of neutrophils in tuberculosis, we focused on the fate of Mtb in neutrophils. We demonstrated that Mtb-infected neutrophils are short-lived and actively engulfed by two ontologically distinct lung macrophages, alveolar and interstitial macrophages in vivo. RNA-seq data also confirms the expression of efferocytosis-related genes in both types of macrophages. Importantly, Mtb remains viable in lung macrophages after being transported from neutrophils, implying a critical step for Mtb spreading between host phagocytes. Furthermore, neutrophils upregulate CD64 and reveal a more activated phenotype. Exploitation of reporter Mtb strains, we observed comparable replication status of Mtb in both CD64+ and CD64− neutrophils, indicating that Mtb replication is independent of the neutrophil activation state. Mechanistically, although CD64+ neutrophils are more glycolytically active than CD64− neutrophils, CD64+ neutrophils highly express CD36, and internalize greater amounts of fatty acid, LDL and oxLDL, the preferred nutrients for Mtb growth. In conclusion, our data support the hypothesis that neutrophils function as a transient carrier that delivers Mtb to macrophages. The metabolic state in neutrophils is distinct from macrophages yet still represents a hospitable environment supporting bacterial growth.
Ephrin B receptor tyrosine kinase ligands modulate the germinal center reaction and control humoral immune responses to malaria
Authors: Olatunde Adesola C , Mimche Patrice N , Seely Spencer O , Stewart Taryn P , Lamb Tracey J
Published: January 2023

Malaria remains a global health priority causing half a million deaths annually. Protective antibody responses that can control the Plasmodium parasites that cause malaria are an essential component of naturally acquired immunity and develop after years of continuous exposure to Plasmodium parasites. The protective humoral response is short-lived and tends to wane in the absence of parasite re-infections. The cellular and molecular mechanisms that prevent the development of long-lived humoral immunity against malaria remain poorly understood. Recently, ephrin B1, a ligand for the Eph B receptor tyrosine kinase subfamily, was shown to be involved in T follicular helper (Tfh) cell recruitment, retention and contact-dependent interaction with germinal center (GC) B cells, key processes in the production of efficacious antibody responses. We hypothesize that EphB/EphrinB signaling pathway is required for the development of Plasmodium humoral responses. Using a non-lethal P. yoelii XNL infection model, we observed an upregulation in the expression of Ephrin B on both GC B cell and Tfh cells at the peak of the infection in wild type mice. Selective deficiency of Ephrin B1/B2 on B cells (CD19cre+ Ephrin B1/B2fl/fl mice) led to lethality in some mice infected with an otherwise non-lethal P. yoelii XNL infection. On the other hand mice that lack the expression of Ephrin B1/B2 on T cells (CD4cre+ Eprhin B1/B2fl/fl mice) were able to control P. yoelii XNL infection. Our data show a requirement for Ephrin B1/B2 signaling on B cells for an effective contact-dependent interaction with Tfh cells and for optimal antibody responses during Plasmodium infection.
Characterization of a potential host-directed therapy against TB in non-human primates
Authors: Winchell Caylin G , Maiello Pauline A. , Flynn JoAnne L
Published: January 2023

An estimated 558,000 cases of drug-resistant tuberculosis (TB) occurred in 2017, emphasizing a need for new treatment strategies. We assessed a host-directed therapy (HDT) for TB using two FDA-approved drugs. Linezolid (LZD) is a potent antibiotic for antibiotic-resistant Gram-positive infections and is an effective treatment for TB. However, lengthy antibiotic regiments (>4 weeks) may lead to LZD-associated host toxicities, most commonly bone marrow (BM) suppression. LZD toxicities are mediated by IL-1, a pathway important for early immunity during Mtb infection that later contributes to pathological TB disease. We hypothesized LZD efficacy could be enhanced by addition of Anakinra, a recombinant IL-1R antagonist (rIL-1RA), by reducing BM toxicity and TB-associated inflammation. We tested this HDT in a non-human primate (NHP) model which emulates the spectrum of human TB, combined with PET/CT to monitor disease progression. NHPs with active TB were treated with LZD or LZD+rIL-1RA and tracked by PET/CT to monitor granuloma formation and inflammation. Analysis of BM erythroid progenitor cells determined little evidence of BM suppression associated with short-term high dose LZD. However, we observed sterilization of the majority of granulomas and a trend of enhanced bacterial clearance with rIL-1RA. Addition of rIL-1RA decreased granuloma inflammation (measured by PET/CT) compared to LZD alone, however few differences in immune cell populations were observed. Together, these data indicate LZD as a promising addition to treatment of drug resistant TB and the potential of HDTs in improving therapy.
Mechanisms controlling Cryptococcus Intracellular Parasitism
Authors: de Figueiredo Paul , Pandey Aseem , Ding Sheng-Li , Qin Qing-Ming , Gupta Rahul , Gomez Gabriel , Lin Furong , Feng Xuehuan , de Costa Luciana Fachini , Chaki Sankar P , Katepalli Madhu , Case Elizabeth , Van Schaik Erin , Sidiq Tabasum , Khalaf Omar , Arenas Angela , Kobayashi Koichi S , Samuel James E , Rivera Gonzalo , Alaniz Robert Christopher , Sze Sing-Hoi , Qian Xiaoning , Brown William J , Rice-Ficht Allison , Russell William , Ficht Thomas A
Published: January 2023

Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we describe insights that have emerged from a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis revealed diverse host proteins that were differentially phosphorylated following fungal infection, indicating global reprogramming of host kinase signaling during this process. Notably, phagocytosis of the pathogen activated the host autophagy initiation complex (AIC) as well as regulatory components that reside upstream of this complex. Cn-containing vacuoles (CnCVs) were found to be decorated with the cell surface marker CD44, which colocalized with components of the AIC complex. Taken together, these findings suggest that associations between CD44 and AIC proteins confer susceptibility to infection, thereby implicating novel host mechanisms in regulating fungal intracellular parasitism.
β-glucan priming enhances neutrophil Fcγ receptor-mediated antifungal activity during oral mucosal infection
Authors: Swidergall Marc , Solis Norma V , Wang Zeping , Phan Quynh T , Marshall Michaela E , Lionakis Michail S , Pearlman Eric , Filler Scott G
Published: January 2023

Neutrophils are key to control fungal proliferation in the oral cavity. Recently we identified that the ephrin type-A receptor 2 (EphA2) functions as an oral epithelial cell β-glucan receptor that triggers epithelial cell production of proinflammatory mediators in response to Candida albicans infection. Because EphA2 is also expressed by neutrophils, we investigated the role of EphA2 in neutrophil candidacidal activity during OPC. We found that when OPC was induced in EphA2−/− mice, there was delayed accumulation of phagocytes into the oral tissues and more severe disease relative to wild-type mice. Depletion of neutrophils in EphA2−/− mice did not further increase oral fungal burden, nor did adoptive transfer of EphA2−/− neutrophils into CD18−/− mice decrease oral fungal burden, indicating that EphA2 on neutrophils plays a central role in the host defense against OPC. Mice lacking the Fcγ receptor I (CD64) were also highly susceptible to OPC. In vitro studies showed that EphA2 signaling augments neutrophil Fcγ receptor-mediated antifungal activity. The interaction of serum-opsonized C. albicansyeast with neutrophil EphA2 activated the MEK-ERK signaling pathway, leading to NADPH subunit p47phox site specific phospho-priming. EphA2-induced priming of neutrophil p47phox increased intracellular ROS production and enhanced killing of opsonized C. albicans yeast. Thus, in neutrophils, EphA2 serves as a receptor for β-glucans that enhances Fcγ receptor-mediated antifungal activity and is crucial to control early fungal proliferation during OPC.
The role of NKT cells in systemic Methicillin-resistant Staphylococcus aureus (MRSA) infection
Authors: Genardi Samantha , Cao Liang , Visvabharathy Lavanya , Berdyshev Evgeny , Wang Chyung-Ru
Published: January 2023

Methicillin-resistant Staphylococcus aureus (MRSA) is an extracellular pathogen responsible for many deaths in the hospital setting each year. The role of conventional T cells in MRSA infection has been extensively studied, however the role of CD1d-restricted natural killer T (NKT) cells in MRSA infection is unknown. NKT cells are divided into 2 subsets, type I expressing an invariant TCR, and type II NKT cells, expressing diverse TCR. Mice lacking both NKT cell subsets showed increased bacterial burdens, inflammatory foci, and neutrophilic infiltrates in the kidneys and increased bacterial burdens in the liver at early times post infection. Both type I and type II NKT cells are activated after SA infection and produce IFN-g. We identified polar lipid species from the cell membrane of MRSA that induce CD1d-dependent IFN-g production by type II NKT cells. We also demonstrate that type I NKT cells, together with neutrophils, control bacterial burdens in infected macrophages. Therefore, NKT cells can become activated and produce protective cytokines in response to MRSA infection that enhances control of infection prior to conventional T cell activation.
Transcriptional profile of CD8+Vα7.2+CD161+ MAITs reveals distinct gene expression compared to CD8 memory T cells
Authors: Lindestam Arlehamn Cecilia S , Pomaznoy Mikhail , Burel Julie G , Kuan Rebecca , Seumois Gregory , Vijayanand Pandurangan , Lewinsohn David Michael , Sette Alessandro , Peters Bjoern
Published: January 2023

Here, we applied a cell population transcriptomics strategy to sorted human memory CD8 T cells to define novel immune signatures of latent tuberculosis infection (LTBI) and understand the phenotype of tuberculosis (TB)-specific T cells. We found a 41-gene signature that could discriminate between memory CD8 T cells from healthy LTBI subjects and noninfected controls. The gene signature was dominated by genes known to be associated with mucosal associated invariant T cells (MAITs) and reflected the lower frequency of MAITs observed in individuals with LTBI. There was no evidence for a conventional CD8 T cell specific signature between the two cohorts. We therefore investigated the MAITs in more detail in these cohorts. Phenotyping based on Vα7.2 and CD161 expression and MR1 tetramers revealed 2 distinct populations of CD8+Vα7.2+CD161+ T cells: MR1 tetramer-restricted and MR1 tetramer-unrestricted, both of which had a distinct gene expression profile compared to CD8 memory T cells. Transcriptomic analysis of LTBI vs. noninfected individuals did not reveal significant differences for MR1 tetramer-restricted cells. However, interestingly, gene expression of MR1 tetramer-unrestricted cells showed a very different profile with large inter-individual diversity and a TB-specific signature. This was further strengthened by a more diverse TCR-α and -β repertoire of MR1 tetramer-unrestricted cells as compared to MR1 tetramer-restricted. Thus, cell population transcriptomics revealed a dominant MAIT signature in CD8 memory T cells that upon detailed investigation provided novel insights into the phenotype of different MAIT populations implicated in tuberculosis.
NK cells negatively regulate CD8 T cells to promote immune exhaustion and chronic Toxoplasma gondii infection
Authors: Gigley Jason P. , Ivanova Daria L , Denton Stephen L
Published: January 2023

Previous studies demonstrate CD8 T cell immune exhaustion is a major factor in promoting chronic Toxoplasma gondii (T. gondii) infection. How other immune cells are impacted by chronic T. gondii infection to promote CD8 T cell exhaustion and chronic parasite infection are not known. We investigated natural killer cells (NK cells) and how they contribute to chronic Toxoplasmosis. We find NK cells are not exhausted because NK cell numbers do not decrease during late stage infection and do not up regulate PD-1 or LAG3. NK cells in chronic parasite infection have reduced IFNγ, do not produce IL-10 or increase their expression of PD-L1, however, they expressed elevated CD107a. We also find NK cells become more highly mature (KLRG1+) during chronic T. gondii infection. We conclude that they do not become exhausted as do CD8 T cells, but alter their function during chronic T. gondii infection. Importantly, we demonstrate NK cell depletion with anti-NK1.1 is therapeutic and rescues mice from death normally caused by CD8 T cell exhaustion and parasite reactivation. This treatment reduced CD8 T cell apoptosis, increased parasite specific polyfunctional CD8 T cell responses and prevented parasite reactivation. NKp46+ NK cells were enriched for expression of CD94 and NKG2A. NKp46 ligand and Qa-1b expression were altered to promote enrichment of this specific NK cell population. Blockade with anti-NKp46 also rescued the mice from death associated with exhausted CD8 T cells and parasite reactivation. We hypothesize NK cells via NKp46 significantly contribute to promoting chronic T. gondii infection. Understanding how NK cells develop this response will improve therapies for individuals at risk for reactivation of chronic infections.
CCR5-mediated recruitment of NK cells to the kidney is a critical step for host defense to systemic Candida albicans infection
Authors: Kang Sang W. , Tran Vuvi G. , Nguyen Nu Z. N. , Lee Saerom , Kim Juyang , Kim Hye J. , Lee Jong S. , Cho Hong R. , Kwon Byungsuk
Published: January 2023

CCR5 regulates the trafficking of various immune cells to the site of infection. Expression of CCR5 and its ligands were rapidly increased in the kidney after systemic Candida albicans infection and infected CCR5−/− mice showed increased mortality and morbidity, indicating that CCR5 contributes to an effective defense mechanism against systemic C. albicans infection. The susceptibility of CCR5−/− mice to C. ablicans infection was due to their impaired capacity of fungal clearance, which in turn resulted in exacerbated renal inflammation and damage. CCR5-mediated recruitment of NK cells to the kidney in response to C. albicans infection was necessary for the anti-microbial activity of neutrophils, the main fungicidal effector cells. Mechanistically, C. albicans rapidly induced expression of IL-23 and IL-15 by CD11b+CD11c+ dendritic cells (DCs) and Ly6C+ monocytes, respectively. IL-23 and IL-15 in turn augmented the fungicidal activity of neutrophils through GM-CSF production by NK cells. As GM-CSF potentiated production of IL-23 and IL-5 in response to C. albicans, a positive feedback loop formed between NK cells and DCs or Ly6C+ monocytes seemed to function as a central amplification point for host defense. Taken together, our results suggest that CCR5-mediated recruitment of NK cells to the site of fungal replication is a critical step that underlies innate resistance to systemic C. albicans infection.
Eosinophils in tick transmitted ehrlichial infection
Authors: Saito Tais B , Walker David H
Published: January 2023

Vector-borne diseases pose significant global health concerns with tick-borne disease doubling over the past 13 years. Tick-borne ehrlichiosis are life-threatening diseases that affect humans and animals. Many studies have been focused on the pathogenesis of these infections; however not much is known about in vivo immune responses to tick transmitted ehrlichiosis. With our model of a human pathogen Ehrlichia (EML) transmitted by ticks, we observed intense changes at the site of infection that persisted for several days. Lethal infection suggests an involvement of vascular dysfunction in disease pathogenesis. Therefore, the goal of this study was to evaluate the granulocyte response to tick transmission, primarily eosinophils. Skin site of the arthropod attachment showed intense infiltration of granulocytes, extending through dermal and subcutaneous tissue. The predominant cells contained eosinophilic granules and were surrounded by other poly and mono-nuclear cells. Also, we further identified increased cellular signaling associated with granulocyte recruitment and vascular dysfunction. The progression of disease was also characterized by increased eosinophils in the blood and immune mediators (e.g. IL-1β) in serum, indicative of proliferation, chemotaxis, and activation of these cells. A needle infection model demonstrated that granulocytes were implicated in immunopathology, bacterial replication, over-production of cytokines/chemokines. We believe that tick transmitted ehrlichiosis induces eosinophilic inflammation that result in release of an arsenal of mediators that regulate inflammation and vascular permeability and could be involved in the establishment of infection and/or disease development.
Demonstration of TLR response in O. mykiss by the bacterial 23S rRNA motif
Authors: Head Kelly , Kobayashi Koichi S , Criscitiello Michael F , Vijayan Saptha
Published: January 2023

The innate immune system is designed to recognize conserved pathogenic sequences known as pathogen-associated molecular patterns (PAMPs), and it is through the modification of these PAMPs that pathogens can subvert innate immune mechanisms. The target of this study is a conserved motif in the bacterial 23S rRNA, which demonstrates a reduced ability to mutate due to lethality to the organism from degraded ribosomal function. The detection of this motif through the pattern recognition receptor (PRR) TLR13 was demonstrated to elicit an immune response across eight animal phyla and Protistan amoeba phyla through this receptor or its orthologs. In teleost fish, the representative model rainbow trout (Oncorhynchus mykiss) was used to study 23S rRNA recognition by TLR22 and TLR13 initiation of an immune response.
mTORC1-mediated macrophage polarization into M1 Contributes to Ehrlichia-induced sepsis
Authors: El Andaloussi Abdeljabar , Haloul Mohamed Abdalla , Kader Muhamuda , Tominello Tyler , Wells Jakob Z. , Ismail Nahed
Published: January 2023

Macrophages heterogeneity that undergo dynamic changes in phenotype and function as well as reprograming of immune response to multiple intracellular bacterial infections is an important key to understand the host responses to infection. Ehrlichia is an obligate Gram-negative intracellular bacterium that cause potentially fatal human monocytotropic ehrlichiosis (HME). We utilized murine models of mild and fatal ehrlichiosis caused by Ehrlichia muris and Ixodes Ovatus Ehrlichia (IOE), respectively, to evaluate the macrophages polarization to inflammatory (M1) and regenerative (M2). Our data show that in vivo and in vitro infections of macrophages with IOE significantly polarize macrophages towards M1, while EM infection enhances M2 polarization. M1 polarization of macrophages in IOE-infected mice was associated with recruitment and/or expansion of granzyme B+ neutrophils, NK cells, IL-17+ NKT cells, and IL-17+ CD4 Th17 cells compared to EM infection. We previously identified mTORC1 (mammalian target of rapamycin complex-1), a negative regulator of autophagy and the key mediator for immunopathology during IOE infection. We found that M1 polarization in the livers of IOE-infected mice is dependent on mTORC1 activation. Blockade of mTORC1 activation with rapamycin decreased frequency of Th17, and enhanced autophagy, which in turn decreased inflammation as well as the pathogenic immune response during lethal IOE infection. Together, these data reveal a key role of mTORC1 in M1 polarization of macrophages, which likely promotes Ehrlichia-induced sepsis. Understanding the dynamic changes of macrophage polarization and how it affects immune responses to infection is crucial for development novel therapeutic strategies.
Identification and characterization of Escherichia coli genes associated with grazing resistance to the social amoeba Dictyostelium discoideum
Authors: Snyder Michelle , Perez-Vazquez Marta , Johnson De’sha , Rasko David A.
Published: January 2023

Pathogenic Escherichia coli strains remain a significant health challenge, particularly in developing countries. The social amoeba, Dictyostelium discoideum, which phagocytizes bacteria for nutritional purposes, serves as a host model for several pathogenic bacteria, including E. coli. Here we have characterized the interactions of D. discoideum with 35 representative E. coli strains, and found that strains sensitive and resistant to D. discoideum grazing were found in each E. coli pathotype. Whole genome phylogenomic analysis revealed that grazing resistance is distributed among the phylotypes, although resistant strains are enriched in the B2 phylogenomic group. Comparative genomic analysis identified genes more prevalent among strains resistant to D. discoideum grazing, including genes associated with adhesion, carbohydrate modification, iron transport, signaling and metabolism. Interestingly, the gipA (growth in Peyer’s Patches) gene, which has been found associated with microbiota in patients with Crohn’s disease, was widely present in E. coli strains resistant for D. discoideum grazing. We are currently working to characterize the role that E. coli genes that have been found associated with grazing-resistance might play in resistance to phagocytosis by D. discoideum, through creation and analysis of E. coli strains manipulated for expression of the identified genes. Such characterization is aimed at providing insight into potentially novel E. coli virulence factors.
12/15-lipoxygenase supports inflammatory cytokine production and PMN recruitment during invasive aspergillosis
Authors: Mackel Joseph J , Jones MaryJane , Steele Chad
Published: January 2023

Eicosanoids are a family of inflammatory lipid mediators produced via the metabolism of arachidonic acid by a network of biosynthetic enzymes. We and others have recently shown that pathways within this network, specifically the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) pathways, participate in induction of protective immune responses during invasive aspergillosis (IA). Based on the high degree of cooperation and interconnection within the eicosanoid network, we hypothesized that a third core enzyme, 12/15-lipoxygenase (12/15-LOX), also may support innate antifungal responses. We report here that mice deficient in the gene encoding 12/15-LOX, Alox15, exhibit decreased survival and increased fungal burden in a model of IA. These defects were associated with decreased PMN numbers in the lung and decreased levels of PMN chemokines (CXCL1, CXCL2, CCL3) in Alox15−/− mice, while fungal killing by PMNs in vitro was unaffected. Alox15−/− mice produced lower levels of the inflammatory cytokines IL-17A, IL-22, IL-1α and IL-6 during infection. iNKT cells, which we have reported to be a major cell source of IL-22 during IA, were present in decreased numbers in Alox15−/− mice. This reduction was associated with lower levels of IL-7, a cytokine we have recently shown to be required for optimal numbers of iNKT cells during IA. Decreased IL-22 levels were further associated with decreased levels of IL-23. Together, these data indicate that 12/15-LOX is a critical player in induction of multiple aspects of the innate immune response to A. fumigatus.
Role of the protein CagA from Helicobacter pylori in the modifications of the human B lymphocyte cytoskeleton
Authors: Cervantes Rodrigo , Piedra-Quintero Zayda , Sandoval-Montes Claudia , Amieva Manuel , Torres-López Javier , Santos-Argumedo Leopoldo , Maravillas-Montero José Luis
Published: January 2023

The cytotoxin-associated gen A (CagA) is an oncogenic virulence factor of Helicobacter pylori that is related with the development of gastric cancer and gastric MALToma: a B cell non-Hodgkin’s lymphoma.

CagA has an important role in the development of cancer due to its promiscuous interaction with different signaling-mediators. The association of CagA with cytoskeleton regulators generates an effect that gives invasive characteristics to epithelial cells, thus explaining the evolution to late stages of the gastric carcinoma. Although the translocation of CagA has been demonstrated in B lymphocytes, its impact on MALToma development and its effects over B cell cytoskeleton have not been characterized.

Human Raji B cells were transiently transfected to express an EGFP-tagged CagA fusion protein. In culture, these cells displayed altered morphological and adhesion properties when compared with control cells. Fibronectin induced-spreading and adhesion assays were performed to assess the changes observed. The transfected cells exhibited atypical size and shape descriptors, determined by analyses of confocal microscopy images. Concomitantly, the adhesion of CagA+ Raji cells was observed reduced. These phenotypes could be explained by the diminished basal levels of ezrin that we detect in the CagA+ cells, determined by Western blot, that contrast with a high expression of this actin-regulatory molecule in Raji control cells.

Taken together, our results show that the expression of CagA produces important changes in B cell morphology as well as cell adhesion due to alteration in actin cytoskeleton regulation; changes that could explain an invasive and aggressive B lymphocyte profile.
Lymphotoxin β receptor: A crucial role in Toxoplasma gondii infection
Authors: Wichert Anne , Sorg Ursula , Degrandi Daniel , Pfeffer Klaus
Published: January 2023

After infection with the obligate intracellular protozoan parasite Toxoplasma gondii (T. gondii) the production of cytokines (especially Interferon γ (IFNγ)) induces potent cell autonomous effector mechanisms which can control the pathogen. Lymphotoxin β receptor (LTβR) mediated signalling plays an important role in the efficient initiation of innate and adaptive host responses to a variety of pathogens.

Therefore, the immune response to T. gondii infection was analyzed in vivo and in vitro in LTβR deficient (LTβR−/−) and wildtype mice (WT) mice. Compared to WT mice, LTβR−/− mice showed an increased parasite burden and a markedly increased mortality in the acute phase of T. gondii infection. FACS analysis revealed that while these mice are generally able to generate T. gondii specific CD8+ T cells this does not seem sufficient for clearing the infection. Tissue and serum of LTβR−/− mice revealed deregulated cytokine expression patterns and production, particularly regarding interferons and interleukins. Since IFNγ mediated upregulation of murine guanylate-binding proteins (mGBPs) is essential for parasite clearance, expression and localization of mGBPs was evaluated. In vivo, delayed/reduced up-regulation of mGBPs expression was observed, whereas initial in vitro experiments in LTβR−/− fibroblasts demonstrated that mGBPs can be upregulated after IFNγ treatment and are able to localize at the parasitophorous vacuole.

These data suggest that defects in cytokine signalling, especially IFNγ deregulation, may contribute to the decreased survival rates of LTβR−/− mice in T. gondii infection. New insights into the pathology of T. gondii could provide new therapeutic strategies for the treatment of human toxoplasmosis.
Effect of Sodium Butyrate Supplementation on the Gut Microbiome during Colorectal Cancer
Authors: Alrafas Haider Rasheed Daham , Busbee Brandon P. , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Colorectal Cancer(CRC) is most common cancer of gastrointestinal tract. In the current study, we investigated the effects of sodium butyrate (BUT) on azoxymethane-dextran sodium sulfate (AOM-DSS)-induced CRC murine model. The data shows that supplementation with BUT reduces overall CRC severity. Endoscopic and histopathological evaluation showed BUT decreased colonic tissue damage and tumor growth. In addition, BUT was able to decrease myeloid-derived suppressor cells (MDSCs) in the blood and BUT was able to altered T cells populations which included significant increases in Tregs and IL-10 producing T cells while decreasing Th1 and Th17 cells. To investigate alterations in the gut microbiome we performed 16S rRNA sequencing of cecal flushes and validated significant findings. The data showed AOM-DSS administration led to significant decreases in bacterial species Ruminococcus gnavus and Akkermansia muciniphila, however, mice that were treated with BUT showed a reversal in these gut microbial alterations. We quantified short chain fatty acids (SCFA). AOM-DSS administration led to a significant decrease in i-butyric and n-butyric acids, but mice supplemented with BUT restored these SCFA levels significantly. Lastly, we examined in vivo and in vitro expression of histone deacetylases (HDACs) after supplementing BUT. Results showed that BUT was able to decrease Class I (HDAC I) and class II (HDACII) in both in vivo and in vitro studies which correlated with a significant increase in Tregs. Collectively, these data showed that BUT can ameliorate CRC by preventing cancer-associated gut microbial dysbiosis through increase in i-butyric and n-butyric acids level which leads to an anti-inflammatory response by suppressing HDACs.
Cytokine mediated control of microbiota and inflammation in atherosclerosis
Authors: Koltsova Ekaterina , Peshkova Iuliia , Dzutsev Amiran , Aghayaev Turan , Hazen Stanley , Trinchieri Giorgio , Fatkhullina Aliia
Published: January 2023

Atherosclerosis is lipid-driven, chronic inflammatory disease of the arterial wall. While commensal microbiota is involved in the distal regulation of systemic immune responses, how this distant connection influences the development of atherosclerosis and what are the underlying mechanisms remains largely unknown. In a mouse model of atherosclerosis, we found that disease was augmented when expression of the otherwise inflammatory cytokine IL23 was ablated. IL23 and its immediate downstream target IL22 restrict atherosclerosis by preventing outgrowth of microbiota, as inactivation of IL23/IL22 signaling led to dysbiosis and expansion of bacteria with pro-atherogenic properties, due to defective production of antimicrobial peptides in the intestine. These pro-atherogenic bacteria contributed to elevated serum levels of several pro-atherogenic metabolites, which in turn induced osteopontin (OPN) expression by subsets of myeloid cells including aortic macrophages. Microbiota transfer from IL23 deficient mice accelerated atherosclerosis, while microbial depletion or IL22 administration reduced aortic osteopontin expression and ameliorated the disease. Overall, our work uncovers the innate inflammatory IL23-IL22 cytokine signaling axis as a key regulator of atherosclerosis that controls diet-induced expansion of pro-atherogenic microbiota, and argues for informed usage of cytokine blockers with regard to cardiovascular side effects driven by microbiota and inflammation
The role of oral pathobionts in the pathogenesis of gastrointestinal inflammation
Authors: Kamada Nobuhiko
Published: January 2023

Mounting evidence suggests that gut microbiota contributes to the pathogenesis of inflammatory bowel disease (IBD). However, the identity of disease-associated pathobionts is unknown. Recent studies showed that patients with IBD are colonized by atypical bacterial members, particularly those residing in the oral cavity (e.g., Enterobacteriaceae, Pasteurellaceae, Fusobacteriaceae, and Veillonellaceae). Given that patients with IBD often accompanied by oral inflammation and/or oral dysbiosis, it is conceivable that oral disease (inflammation, dysbiosis) may in turn impact on the development of intestinal pathology. In this study, we demonstrate that periodontal inflammation exacerbates colitis in mice. Periodontitis in mice was induced by insertion of the oral ligature. Colitis was induced by the administration of dextran sulfate sodium (DSS). Mice with periodontitis developed more severe DSS-induced colitis compared to those without periodontitis. During periodontitis, Enterobacteriaceae were expanded in the oral cavity. Amassed Enterobacteriaceae of oral origin were then ingested and ectopically colonized the colon. Gut colonization by the oral Enterobacteriaceae elicits the activation of the inflammasome and promotes inflammation. Taken together, inflammation in the oral mucosa may foster the abnormal expansion of oral pathobionts whose ectopic gut colonization aggravates IBD.
Toll-like Receptor 2/IL-1β signaling is required for sensing and responding to a commensal bacterium at the ocular surface
Authors: Zhu Wenjie , St. Leger Anthony , Almaghrabi Fatimah , Xu Xiaoyan , Caspi Rachel
Published: January 2023

Conjunctiva-associated lymphoid tissue (CALT) resembles a conventional mucosal immune tissue, and harbors a variety of immune cells. CALT is exposed to diverse microorganisms that come in contact with the ocular surface. Previously, we demonstrated that colonization of the ocular mucosa with the commensal bacterium, Corynebacterium mastitidis (C. mast), results in increased resistance of the ocular surface to infectious fungal and bacterial pathogens. Further, we showed that this effect is due to IL-17 produced by Vγ4+γδ T cells that respond to C. mast in an IL-1-dependent fashion. In this study, we show that the toll-like receptor 2 (TLR2) and IL-1β are important for IL-17 production, activation and proliferation of Vg4+ γδ T cells. Using in vivo and in vitro approaches, we show that cells of TLR2 KO mice have reduced production of IL-1β in response to C. mast. These mice also fail to recruit Vg4+γδ T cells and neutrophils to the conjunctiva. We interpret these data to mean that TLR2 is required for sensing this commensal on the conjunctiva and its draining lymph nodes to activate the inflammasome and consequent IL-1 production, which in turn drives the IL-17 response. Our data highlight the importance and the role of the innate receptor TLR2 in the generation of the ocular mucosal immune response and in maintenance of the homeostatic γδ T cell response at the ocular surface.
Gut antibody deficiency in CD19−/− mice results in a spontaneous Celiac-Like sprue
Authors: Mohammed Ahmed Dawood , Kubinak Jason L.
Published: January 2023

Primary immunodeficiencies are heritable disorders of the immune response that most commonly manifest as an inability to generate immunoglobulin A (IgA). Despite its prevalence, IgA deficiency is considered a benign form of antibody deficiency due to a lack of easily observed clinical symptoms. CD19 is an important co-receptor that facilitates B cell receptor signaling and B cell development into antibody-secreting plasma cells. CD19 deficiency is a known genetic risk factor for the development of common variable immunodeficiency (CVID). Here, we utilize the CD19−/− mouse model to quantify the role of IgA deficiency on gut homeostasis. Our results indicate that CD19−/− mice develop a severe gut IgA deficiency associated with aberrant anti-commensal IgA responses that result in the outgrowth of anaerobic bacteria in the gut. More importantly, anaerobic expansion results in intestinal malabsorption under steady-state conditions. Intestinal malabsorption is associated with defects in normal hepatobiliary circulation, vitamin deficiency, and dyslipidemia. Additionally, results from histological analysis and RNAseq experiments indicate that CD19−/− mice develop intestinal epithelial cell (IEL) lymphocytosis mediated by CD8aa+TCRgd lymphocytes. Finally, CD19−/− mice, while deficient in anti-gliadin antibody (AGA) production compared to WT mice, demonstrate a significantly stronger relationship between serum AGA titers and total serum IgG and IgA production compared to WT animals and have significantly elevated ileal expression of tissue transglutaminase 2. Collectively, we believe that we have identified a novel mouse model of sero-negative celiac disease that will be of great value to the celiac research community.
Surface and cytoplasmic tumor cell PD-L1 differentially mediate virulence in ovarian cancer and melanoma through mTOR activation
Authors: Hambright Heather Graham , Gupta Harshita B. , Padron Alvaro S , Vadlamudi Ratna , Chen Yidong , Osmulski Pawel A , Curiel Tyler J
Published: January 2023

Tumors express intracellular PD-L1 that we showed increases ovarian cancer (OC) and melanoma virulence by increasing mTORC1 and tumor stem cells (TSC). We made PD-L1KO(CRISPR) in ES2 human OC and B16 melanoma cells and re-expressed PD-L1 only on cell surface (PD-L1surf) or in cytoplasm (PD-L1cyto) with specific expression vectors. Subcellular PD-L1 location in all clones was validated by immunoblots, confocal microscopy, and co-IP/mass spec. We selected clones with PD-L1 surf or cyto expression similar to parental cells. PD-L1KO reduced cell diameter 30% which was restored partially in PD-L1surf and fully in PD-L1cyto in ES2 and B16. PD-L1KO significantly decreased viability in all cell lines tested which was fully rescued in PD-L1surf and PD-L1cyto. PD-L1surf ES2 and B16 grew slower in vitro and in vivo than PD-L1cyto and only PD-L1cyto rescued F-actin polymerization phenotype seen in PD-L1KO ES2 and B16. RNA-seq showed that surf and cyto PD-L1 differentially altered canonical signaling pathways, notably mTOR, stemness and gene regulation. PD-L1cyto grew much slower than PD-L1surf B16 in NSG mice but generated more TSC in vitro, and PD-L1surf but not PD-L1cyto B16 responded to αPD-L1 in WT. PD-L1cyto but not PD-L1surf induced mTORC1 activation (p-rpS6, p-P70S6K) through mTORC1 lysosomal docking through LAMTOR1/2 seen by confocal and confirmed by co-IP in ES2 and B16. Imaging CyTOF of human melanomas showed PD-L1cyto association with activated mTOR. Thus, surf versus cyto PD-L1 differentially affects tumor biology, signaling, pathology, TIL and treatment response in OC and melanoma. We define a mechanism for PD-L1-driven mTORC1 activation. Surface vs. cytoplasmic PD-L1 and/or mTORC1 could be response biomarkers for αPD-L1 or αPD-1.
Randomized Controlled Phase 2a Study Results Using Anti IL-33 in Food Allergy
Authors: Liu Cherie , Chinthrajah Sharon , Long Andrew , Lyu Shu-Chen , Londei Marco , Nadeau Kari Christine
Published: January 2023

Objectives of this placebo-controlled study were to assess the safety and tolerability of single dose ANB020 administration in peanut-allergic adult patients and to measure the response during a standardized oral food challenge (OFC) after ANB020 or placebo administration compared to baseline peanut food challenge. On Day 1, fifteen highly allergic adult patients were administered 300 milligrams of ANB020 by IV infusion, and five patients were administered placebo injections. To measure patient response, patients were administered ex-vivo whole blood peanut antigen challenges on Day 2 and Day 5 and were administered OFCs on Day 15 and Day 45. The clinical outcomes and cellular responses of patients were analyzed. For patients given ANB020, 0% at Baseline, 67% on Day 15, and 57% on Day 45 passed the OFC; no placebo patients passed the OFC at Baseline, Day 15, or Day 45. Patients given ANB020 also experienced fewer adverse events (abdominal pain, nasal congestion), compared to placebo patients (abdominal pain, vomiting, nausea, asthma, allergic reaction). The median percentages of IL-4 and IL-9 cells in CD69 T cells were lower for patients administered ANB020 compared to patients administered placebo at Baseline, Day 2, and Day 5, and higher at Day 15. Peanut-specific and histamine skin test wheal sizes among all patients stayed consistent, except at Day 15 for patients given ANB020, where both wheal sizes decreased significantly to less than 5 mm. Volumes of peanut-specific IgE and total IgE were smaller for patients given ANB020 at Baseline, Day 15, and Day 45. Thus, study results show ANB020 injections to be potentially helpful in increasing patient tolerability and safety during OFCs.
Prophylactic immunization prevents antiviral T cell subversion by a rat hepatitis C-like virus
Authors: Hartlage Alex , Murthy Satyapramod Srinivasa , Kumar Arvind , Trivedi Sheetal , Dravid Piyush , Sharma Himanshu , Walker Christopher M , Kapoor Amit
Published: January 2023

The hepatitis C virus (HCV) is a leading cause of severe progressive liver diseases by virtue of its ability to evade host immunity and establish lifelong persistence in majority of infected individuals. Despite significant advancements in antiviral therapy, a vaccine to prevent HCV persistence and associated disease is not yet available, in part because there is no small animal model to experimentally dissect mechanisms of immune protection and viral subversion. Here, we used a novel rat model of HCV-related hepacivirus infection to define antiviral T cell immunity during viral persistence and after prophylactic vaccination. Challenge of lab rats with a rodent hepacivirus led to universal persistence characterized by minimal viral diversification and an absence of functional CD8+ T cell responses. Direct visualization of virus-specific CD8+ T cells with a class I tetramer revealed a premature contraction of responses prior to acquisition of antiviral effector functions. By contrast, immunization of rats with a recombinant adenovirus expressing hepacivirus non-structural proteins induced functional CD4+ and CD8+ T cell immunity and significantly prevented hepacivirus persistence in majority of rats after challenge. Loss of vaccine-conferred immunity and subsequent persistence in a single rat was associated with selection of MHC class I escape variants. Transient depletion of CD8+ cells from vaccinated animals prolonged viral replication after challenge, while CD4+ cell depletion led to virus persistence. These data provide direct in vivo evidence that T cells are important for resolution of persistent hepacivirus infection and that subversion of the response can be successfully prevented by prophylactic immunization.
Unraveling the mechanism of type-1 immunity induction by defective viral genomederived oligonucleotides
Authors: Fisher Devin G , Lopez Carolina B
Published: January 2023

Many newly developed vaccines use killed pathogens or pathogen subunits as antigens. Unfortunately, these inert antigens do not contain danger signals required to induce robust adaptive immunity. Adjuvants are added to vaccines to provide danger signals that help boost and shape the immune response. Currently, no approved adjuvants can effectively skew the immune response towards type-1 immunity including CD8+ T cells necessary for the clearance of viruses and other intracellular pathogens. Most viruses induce type-1 immunity and this process can be leveraged to generate type-1 immunity inducing adjuvants. The primary immunostimulatory molecules during paramyxovirus infections are defective viral genomes (DVGs). We identified the immunostimulatory motif of a Sendai virus DVG and created a DVG-derived oligonucleotide (DDO) to test as an adjuvant. Intramuscular injection of DDO with inactivated influenza virus induced robust protective type-1 immunity characterized by CD8+ T-cell responses and antibodies of the IgG2c isotype. This immune response was dependent on type I interferon signaling. Mice lacking MyD88, the adaptor for TLR and IL-1 signaling, did not produce type-1 humoral or cellular responses in response to a DDO-adjuvanted vaccine. Current studies focus on further understanding the molecular and cellular mechanisms mediating the type I interferon-dependent response to DDOs. This knowledge may allow for directed vaccine design for intracellular pathogens.
Antigen-specific CD8 T cell response following genital chlamydial challenge is a predictive biomarker of vaccine efficacy in protection against reproductive tract pathology
Authors: Murthy Ashlesh K , Warda Katerina , Moy Lindsay , Musunuri Tarakarama , Bui Nhi , Wright-McAfee Erika , Ramsey Kyle H , Li Weidang
Published: January 2023

TNF-α producing antigen (Ag)-specific CD8 T cells induce upper genital tract (UGT) pathology following primary C. muridarum infection; therefore, we hypothesized that a vaccine regimen must subvert Ag-specific CD8 T cell response following immunization, and/or challenge to attenuate Chlamydia-induced UGT pathology. We evaluated three immunization regimens: live-chlamydial elementary body intranasal (EB-i.n.), live-EB i.m. (EB-i. m.), and irrelevant antigen bovine serum albumin i.n. (BSA-i.n.) that induced near-total (90%), partial (50%), and no (0% ) protection, respectively against oviduct pathology following i.vag. C. muridarum challenge in C57BL6/J animals. Live-EB-specific IFN-γ, TNF-α, and IL-17 response from purified splenic CD8 T cells was evaluated on days 7, 14, and 21 after immunization. There was no detectable reduction of Ag-specific CD8 T cell response following immunization with protective vaccine regimens. We evaluated splenic live-EB-specific CD8 T cell responses on days 3, 6, 9, and 12 following i.vag. chlamydial challenge. All groups of challenged animals displayed comparable induction of Ag-specific CD8 T cell cytokine responses on day 6 after challenge. As early as day 9 after challenge, Ag-specific IFN-γ and TNF-α production from CD8 T cells was significantly reduced in EB-i.n.-, compared to the EB-i.m.- or BSA-i.n.-immunized animals. On day 12, both EB-i.n.- and EB-i.m.-immunized animals displayed minimal Ag-specific TNF-α production, compared to enhanced TNF-α production from CD8 T cells in BSA-i.n. immunized animals. These results suggest that TNF-α producing CD8 T cell response could serve as a predictive biomarker of anti-chlamydia vaccine efficacy against reproductive pathology.