Portal Of Entry For Pathogens Research Articles

Coral Disease and Ingestion: Investigating the Role of Heterotrophy in the Transmission of Pathogenic Vibrio spp. using a Sea Anemone (Exaiptasia pallida) Model System.

Understanding disease transmission in corals can be complicated given the intricacy of the holobiont and difficulties associated with ex situ coral cultivation. As a result, most of the established transmission pathways for coral disease are associated with perturbance (i.e., damage) rather than evasion of immune defenses. Here, we investigate ingestion as a potential pathway for the transmission of coral pathogens that evades the mucus membrane. Using sea anemones (Exaiptasia pallida) and brine shrimp (Artemia sp.) to model coral feeding, we tracked the acquisition of the putative pathogens, Vibrio alginolyticus, V. harveyi, and V. mediterranei using GFP-tagged strains. Vibrio sp. were provided to anemones using 3 experimental exposures (i) direct water exposure alone, (ii) water exposure in the presence of a food source (non-spiked Artemia), and (iii) through a "spiked" food source (Vibrio-colonized Artemia) created by exposing Artemia cultures to GFP-Vibrio via the ambient water overnight. Following a 3 h feeding/exposure duration, the level of acquired GFP-Vibrio was quantified from anemone tissue homogenate. Ingestion of spiked Artemia resulted in a significantly greater burden of GFP-Vibrio equating to an 830-fold, 3,108-fold, and 435-fold increase in CFU mL-1 when compared to water exposed trials and a 207-fold, 62-fold, and 27-fold increase in CFU mL-1 compared to water exposed with food trials for V. alginolyticus, V. harveyi, and V. mediterranei, respectively. These data suggest that ingestion can facilitate delivery of an elevated dose of pathogenic bacteria in cnidarians and may describe an important portal of entry for pathogens in the absence of perturbing conditions. IMPORTANCE The front line of pathogen defense in corals is the mucus membrane. This membrane coats the surface body wall creating a semi-impermeable layer that inhibits pathogen entry from the ambient water both physically and biologically through mutualistic antagonism from resident mucus microbes. To date, much of the coral disease transmission research has been focused on mechanisms associated with perturbance of this membrane such as direct contact, vector lesions (predation/biting), and waterborne exposure through preexisting lesions. The present research describes a potential transmission pathway that evades the defenses provided by this membrane allowing unencumbered entry of bacteria as in association with food. This pathway may explain an important portal of entry for emergence of idiopathic infections in otherwise healthy corals and can be used to improve management practices for coral conservation.

Skin immunity during pregnancy

Abstract Pregnancy presents a challenging immunological dilemma. The maternal immune system must tolerate fetal antigens, which implies pregnancy may induce specific mechanisms of immunoregulation. Yet within this regulatory milieu, protection against pathogens must be maintained. How does the maternal immune system navigate these opposing demands? Successfully navigating this immunological dilemma is essential for supporting the reproductive fitness of the organism, and generally, the survival of any mammalian species. Of particular importance in this context are barrier tissues, like the skin, which represent the primary portal of entry for pathogens. These barrier tissues are an especially important first line of defense during pregnancy, since adaptive immunity must be regulated to tolerate the fetal antigens which distribute throughout maternal tissues. However, little is known about how immunity in barrier tissues is altered during pregnancy. Our preliminary studies have identified an increase in ceramide levels in the skin during pregnancy. Ceramides have been shown to be critical mediators of skin barrier function, and may prevent pathogens from breaching the skin. Additionally, in the setting of S. aureus infection, pregnant skin demonstrated increased infiltration of innate immune cells, as well as an increase in type I immunity among adaptive cells. Together, these data suggest that the protective mechanisms of the skin are altered during pregnancy. Further insights in this area may elucidate an especially critical role for barrier tissue immunity in defending against infection during pregancy, and may eventually help guide improved clinical strategies to prevent and treat infections in pregnant mothers. Supported by Perelman School of Medicine's Medical Scientist Training Program and intramural research funding from NIAID.

Prospects for mucosal vaccine: shutting the door on SARS-CoV-2

ABSTRACT The sudden emergence of a highly transmissible and pathogenic coronavirus SARS-CoV-2 in December 2019 from China and its rapid global spread has posed an international health emergency. The rapid development of an effective vaccine is imperative to control the spread of SARS-CoV-2. A number of concurrent efforts to find an effective therapeutic agent or vaccine for COVID-19 (coronavirus disease 2019) are being undertaken globally. Oral and nasal mucosal surfaces serve as the primary portal of entry for pathogens like coronaviruses in the human body. As evidenced by studies on similar coronaviruses (SARS-CoV and MERS-CoV), mucosal vaccination can provide a safe and effective means for the induction of long-lasting systemic and mucosal immunity to confer protection against SARS-CoV-2. This article summarizes the approaches to an effective mucosal vaccine formulation which can be a rewarding approach to combat the unprecedented threat posed by this emerging global pandemic.

Polarized Trout Epithelial Cells Regulate Transepithelial Electrical Resistance, Gene Expression, and the Phosphoproteome in Response to Viral Infection.

The burden of disease is a major challenge in aquaculture production. The fish gill characterized with a large surface area and short route to the bloodstream is a major environmental interface and a significant portal of entry for pathogens. To investigate gill responses to viral infection the salmonid gill cell line RTgill-W1 was stimulated with synthetic dsRNA and the salmonid alphavirus subtype 2 (SAV-2). Epithelial integrity in polarized cells can be measured as transepithelial electrical resistance (TEER) which is defined as the electrical resistance across a cell monolayer. TEER is a widely accepted quantitative measure of cellular integrity of a cell monolayer. TEER increased immediately after stimulation with the synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I:C)). In parallel, tight junction and gene expression of innate immune activation markers was modulated in response to poly(I:C). The SAV-2 virus was found to replicate at a low level in RTgill-W1 cells where TEER was disturbed at an early stage of infection, however, gene expression related to tight junction regulation was not modulated. A strong poly(I:C)-driven antiviral response was observed including increases of Rig-like receptors (RLRs) and interferon stimulating genes (ISGs) mRNAs. At the level of signal transduction, poly(I:C) stimulation was accompanied by the phosphorylation of 671 proteins, of which 390 were activated solely in response to the presence of poly(I:C). According to motif analysis, kinases in this group included MAPKs, Ca2+/calmodulin-dependent kinase (CaMK) and cAMP-dependent protein kinase (PKA), all reported to be activated in response to viral infection in mammals. Results also highlighted an activation of the cytoskeletal organization that could be mediated by members of the integrin family. While further work is needed to validate these results, our data indicate that salmonid gill epithelia has the ability to mount a significant response to viral infection which might be important in disease progression. In vitro cell culture can facilitate both a deeper understanding of the anti-viral response in fish and open novel therapeutic avenues for fish health management in aquaculture.

Prevention of skin damage caused by the protective equipment used to mitigate COVID-19: monthly update.

Prevention of skin damage caused by the protective equipment used to mitigate COVID-19: monthly update.

Impact of multiple hits with cognate antigen on memory CD8+ T-cell fate.

Antigen-driven activation of CD8+ T cells results in the development of a robust anti-pathogen response and ultimately leads to the establishment of long-lived memory T cells. During the primary response, CD8+ T cells interact multiple times with cognate antigen on distinct types of antigen-presenting cells. The timing, location and context of these antigen encounters significantly impact the differentiation programs initiated in the cells. Moderate re-activation in the periphery promotes the establishment of the tissue-resident memory T cells that serve as sentinels at the portal of pathogen entry. Under some circumstances, moderate re-activation of T cells in the periphery can result in the excessive expansion and accumulation of circulatory memory T cells, a process called memory inflation. In contrast, excessive re-activation stimuli generally impede conventional T-cell differentiation programs and can result in T-cell exhaustion. However, these conditions can also elicit a small population of exhausted T cells with a memory-like signature and self-renewal capability that are capable of responding to immunotherapy, and restoration of functional activity. Although it is clear that antigen re-encounter during the primary immune response has a significant impact on memory T-cell development, we still do not understand the molecular details that drive these fate decisions. Here, we review our understanding of how antigen encounters and re-activation events impact the array of memory CD8+ T-cell subsets subsequently generated. Identification of the molecular programs that drive memory T-cell generation will advance the development of new vaccine strategies that elicit high-quality CD8+ T-cell memory.

The Contribution of Pulmonary Dendritic Cells to the Induction and Regulation of T Cell Responses During Respiratory Infection

Abstract The respiratory tract represents a major portal of entry for pathogens, including Group A Streptococci (GAS). GAS represents a significant burden in cases of primary infection as well as cases of secondary bacterial pneumonia following influenza virus infection. Control of infection and eventual resolution is dependent on the local adaptive immune response. This is facilitated by the uptake and subsequent presentation of antigen by dendritic cells (DC) to T cells. Furthermore, after GAS infection, DC drive T cell development towards a Th17 immune response that is associated with pathogen control. Our hypothesis is that pulmonary DC, both resident and recruited, contribute locally to the maintenance and regulation of an effector CD4 T cell response in the context of a GAS infection. Toward this end, our studies are defining the contribution of local DC: T cell interactions in shaping the phenotype and magnitude of Th17 and Tfh responses within the lungs during GAS infection. An improved understanding of the contribution of these DC to the development and regulation of these CD4 T cells responses during GAS infection should elaborate strategies to the development of better therapies.

Airway CD8+CD161++TCRvα7.2+ T Cell Depletion During Untreated HIV Infection Targets CD103 Expressing Cells.

HIV-infected adults are at an increased risk to lower respiratory tract infections (LRTIs). CD8+CD161++TCRvα7.2+ T cells are an innate-like T cell subset that are thought to play an important role in early defense against pathogens in the respiratory tract. HIV infection leads to irreversible depletion of these cells in peripheral blood, however, its impact on this subset in the human airway is still unclear. Here, we show presence of CD103 expressing CD8+CD161++TCRvα7.2+ T cells in the airway that exhibited a distinct cytokine functional profile compared to their CD103− airway counterparts and those from peripheral blood. These CD103 expressing airway CD8+CD161++TCRvα7.2+ T cells were selectively depleted in untreated HIV-infected adults compared to healthy controls. Their frequency was positively correlated with frequency of airway CD4+ T cells. Furthermore, the frequency of airway CD8+CD161++TCRvα7.2+ T cells was also inversely correlated with HIV plasma viral load, while suppressive antiretroviral therapy (ART) resulted in restoration of airway CD8+CD161++TCRvα7.2+ T cells. Our findings show that CD103 expressing airway CD8+CD161++TCRvα7.2+ T cells are functionally distinct and are preferentially depleted during untreated asymptomatic HIV infection. Depletion of CD103 expressing airway CD8+CD161++TCRvα7.2+ T cells, at a major portal of pathogen entry, could partly contribute to the increased propensity for opportunistic LRTIs observed in untreated HIV-infected adults.

Blimp-1 Rather Than Hobit Drives the Formation of Tissue-Resident Memory CD8+ T Cells in the Lungs.

Tissue-resident memory CD8+ T (TRM) cells that develop in the epithelia at portals of pathogen entry are important for improved protection against re-infection. CD8+ TRM cells within the skin and the small intestine are long-lived and maintained independently of circulating memory CD8+ T cells. In contrast to CD8+ TRM cells at these sites, CD8+ TRM cells that arise after influenza virus infection within the lungs display high turnover and require constant recruitment from the circulating memory pool for long-term persistence. The distinct characteristics of CD8+ TRM cell maintenance within the lungs may suggest a unique program of transcriptional regulation of influenza-specific CD8+ TRM cells. We have previously demonstrated that the transcription factors Hobit and Blimp-1 are essential for the formation of CD8+ TRM cells across several tissues, including skin, liver, kidneys, and the small intestine. Here, we addressed the roles of Hobit and Blimp-1 in CD8+ TRM cell differentiation in the lungs after influenza infection using mice deficient for these transcription factors. Hobit was not required for the formation of influenza-specific CD8+ TRM cells in the lungs. In contrast, Blimp-1 was essential for the differentiation of lung CD8+ TRM cells and inhibited the differentiation of central memory CD8+ T (TCM) cells. We conclude that Blimp-1 rather than Hobit mediates the formation of CD8+ TRM cells in the lungs, potentially through control of the lineage choice between TCM and TRM cells during the differentiation of influenza-specific CD8+ T cells.

Metabolic programming of tissue APCs

Inflammation Antigen-presenting cells (APCs) are scattered throughout the body in lymphoid organs and at the portals of pathogen entry, where they act as sentinels of the immune system. Sinclair et al. demonstrate that APCs at different sites have distinctive metabolic signatures and that the development and function of these cells are determined not only by their transcriptional program, but also by their metabolic state (see the Perspective by Wiesner and Klein). The authors identified a central role for mTOR in mediating the metabolic adaptation of such tissue-resident APCs by influencing the immunological character of allergic inflammation. Thus, tissues endow resident APCs with distinctive metabolic characteristics that control APC development and function. Science , this issue p. [1014][1]; see also p. [973][2] [1]: /lookup/doi/10.1126/science.aaj2155 [2]: /lookup/doi/10.1126/science.aao3086

Effectiveness of a multimodal hand hygiene improvement strategy in the emergency department.

Hand hygiene (HH) is essential in preventing nosocomial infection. The emergency department (ED) is an open portal of entry for pathogens into the hospital system, hence the important sentinel function of the ED personnel. The main objective of this study was to assess the effect of a multimodal improvement strategy on hand hygiene compliance in the ED. Our study was a prospective before-and-after study to determine the effect of a multimodal improvement strategy on the compliance of HH in the ED according to the My 5 Moments of Hand Hygiene defined by the World Health Organization. Interventions such as education, reminders, and regular feedback on HH performance and role models were planned during the 3 intervention weeks. In total, 57 ED nurses and ED physicians were observed in this study, and approximately 1,000 opportunities for handrubs were evaluated during the 3 intervention periods. HH compliance increased significantly from baseline from 18% (74/407) to 41% (77/190) after the first intervention and stabilized to 50% (99/200) and 46% (96/210) after the second and third interventions, respectively. Implementing a multimodal HH improvement program significantly improved the HH compliance of ED personnel.

Transcription factors define tissue T cells

Immunology The immune system fights microbial invaders by maintaining multiple lines of defense. For instance, specialized memory T cells [resident memory T cells (Trms)] colonize portals of pathogen entry, such as the skin, lung, and gut, to quickly halt reinfections. Mackay et al. now report that in mice, Trms as well as other tissue-dwelling lymphocyte populations such as natural killer cells share a common transcriptional program driven by the related transcription factors Hobit and Blimp1. Tissue residency and retention of lymphocytes require expression of Hobit and Blimp1, which, among other functions, suppress genes that promote tissue exit. Science , this issue p. [459][1] [1]: /lookup/doi/10.1126/science.aad2035

Hepcidin, Cathelicidin-1 and IL-8 as immunological markers of responsiveness in early developmental stages of rainbow trout

During the early developmental stage of salmonids, high mortality occurs largely as a result of pathogens. These cause low immune competence in fry, producing disease, decreasing production and finally leading to economic losses. Therefore, the aim of this study was to characterise the developmental stages in which rainbow trout acquires immune response capability when challenged with LPS from Pseudomona aeruginosa for 8 h, studying the hepcidin, cathelicidin-1 and IL-8. Total RNA was extracted from fry at 34, 42, 56 and 66 days post hatching (dph). Hepcidin and cathelicidin-1 transcripts were detected only at days 34 and 42, whereas the IL-8 transcript was detected from day 34 to day 66. To analyse the protein expression in the fry, polyclonal anti-peptide antibodies were generated in rabbit. These three immune sera demonstrated the ability to recognise the whole molecule in biological samples. Immunofluorescence showed that skin, gills and intestine mainly responded to the LPS challenge, indicating that these portals of pathogen entry are capturing LPS. This study constitutes a valuable approach, since it has the potential to identify molecules with biological activity that can be used to evaluate the status of fry in culture.

ID: 8: Innate immune defense against influenza virus infection

The mucosal surfaces represent major sites of entry for numerous infectious agents. Consequently, the vast mucosal surfaces are intricately lined with cells and lymphoid organs specialized in providing protective antibody and cellular immunity. Our laboratory’s goal is to understand how immunity is initiated and maintained at the mucosal surfaces that serve as natural portals of entry for pathogens of significant health concerns in the world. In this study, we focused on understanding how viruses are recognized by the innate immune system and how that information is used to generate protective immunity. The myxovirus resistance protein 1 (Mx1) is a dynamin-like GTPase that blocks primary transcription of influenza presumably by binding to viral nucleoproteins. Many inbred laboratory mouse strains have mutations in the Mx1 gene including the C57BL/6 mice. By using mice that have intact Mx1, we demonstrate the critical role of innate sensors in viral control. Further, we reveal a lethal consequence of triggering caspase-1 activation in the absence of innate resistance. Therefore, our results demonstrate that caspase-1 in the absence of antiviral resistance can trigger lethal disease and such conditions may underlie vulnerability to IAV disease in humans.

Goblet cell-associated antigen passages are reduced during enteric infection (MUC4P.831)

Abstract The intestinal mucosa is continually exposed to foreign antigens derived from the diet, commensal bacteria, and pathogens. Regulating the exposure of the immune system to this broad array of antigens is crucial to ensure intestinal homeostasis and to respond appropriately to enteric infections. Using in vivo two-photon imaging of the intestine of live mice, we recently demonstrated that under steady state conditions a subset of small intestinal goblet cells (GCs) transport luminal antigens to CD103+ dendritic cells (DCs) by forming goblet cell-associated antigen passages (GAPs). To understand how enteric infection altered GCs and GAPs, we infected mice with wildtype fluorescently labeled Salmonella typhimurium and evaluated GCs and GAPs using fluorescence microscopy. Within hours of intraluminal exposure, Salmonella preferentially localized around and within GCs that had formed GAPs. 24 hours after oral Salmonella infection, a subset of GCs were found to be apoptotic, and 72 hours after infection, GAPs were significantly decreased beyond the level of GC apoptosis. Salmonella infection resulted in epithelial activation of the epidermal growth factor receptor (EGFR), a potential regulator of GC secretion and GAP formation. These observations indicate that GAPs may provide a portal of entry for pathogens, and that GAP formation is reduced following enteric infection potentially to avoid delivery of luminal antigen to lamina propria immune cells in a hostile environment.

Enhanced Innate Antiviral Gene Expression, IFN-α, and Cytolytic Responses Are Predictive of Mucosal Immune Recovery during Simian Immunodeficiency Virus Infection

The mucosa that lines the respiratory and gastrointestinal (GI) tracts is an important portal of entry for pathogens and provides the first line of innate immune defense against infections. Although an abundance of memory CD4(+) T cells at mucosal sites render them highly susceptible to HIV infection, the gut and not the lung experiences severe and sustained CD4(+) T cell depletion and tissue disruption. We hypothesized that distinct immune responses in the lung and gut during the primary and chronic stages of viral infection contribute to these differences. Using the SIV model of AIDS, we performed a comparative analysis of the molecular and cellular characteristics of host responses in the gut and lung. Our findings showed that both mucosal compartments harbor similar percentages of memory CD4(+) T cells and displayed comparable cytokine (IL-2, IFN-γ, and TNF-α) responses to mitogenic stimulations prior to infection. However, despite similar viral replication and CD4(+) T cell depletion during primary SIV infection, CD4(+) T cell restoration kinetics in the lung and gut diverged during acute viral infection. The CD4(+) T cells rebounded or were preserved in the lung mucosa during chronic viral infection, which correlated with heightened induction of type I IFN signaling molecules and innate viral restriction factors. In contrast, the lack of CD4(+) T cell restoration in the gut was associated with dampened immune responses and diminished expression of viral restriction factors. Thus, unique immune mechanisms contribute to the differential response and protection of pulmonary versus GI mucosa and can be leveraged to enhance mucosal recovery.

Comparison between intestinal and non-mucosal immune functions of rainbow trout, Oncorhynchus mykiss

Since mucosal surfaces represent major portals of entry for pathogens, its associated immune system is important to protect the organism. In this paper, we compared at the cellular and molecular levels intestinal leukocyte suspensions with their head kidney (HK) or peripheral blood (PBL) counterparts to highlight characteristics of intestinal immune functions in healthy rainbow trout. These studies show that intestinal phagocytes are less activated by yeast cells but when they are activated they can ingest as many yeast cells as their HK counterparts. A natural cytotoxic activity could be detected which is twice higher in intestinal than in HK leukocyte preparations. This natural cytotoxic activity is correlated with the expression of transcripts encoding the natural killer enhancement factor (NKEF). Intestinal leukocytes did not respond to an in vitro mitogenic stimulation performed under classical culture conditions. And finally, a high expression of CD8α transcripts was observed in gut leukocyte preparations, suggesting that the intestine could contain a high proportion of T cells expressing the αα homodimeric form of CD8. This kind of comparison on nonimmunized fish provides better knowledge on basal immune functions in the intestine to, analyze later on, immune responses induced by an antigenic stimulation.

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.

Combined mTOR Inhibition and OX40 Agonism Enhances CD8+ T Cell Memory and Protective Immunity Produced by Recombinant Adenovirus Vaccines

The memory CD8(+) T cell population elicited by immunization with recombinant human adenovirus serotype 5 (rHuAd5) vaccines is composed primarily of effector and effector memory cells (T(EM)) with limited polyfunctionality. In this study, we investigated whether treatment with immunomodulators could enhance and/or redistribute the CD8(+) memory population elicited by rHuAd5. Vaccination in combination with both rapamycin (to modulate differentiation) and an OX40 agonist (to enhance costimulation) increased both the quantity and polyfunctionality of the CD8(+) memory T cell population, with expansion of the T(EM) and memory precursor populations. Furthermore, this intervention enhanced protection against multiple virus challenges. Attenuation of adenovirus transgene expression was required to enable the combination rapamycin + OX40 agonist immunomodulatory treatment to further enhance skewing towards central memory formation, indicating that persistence of antigen expression ultimately limits development of this memory population following rHuAd5 immunization. These results demonstrate that during the expansion phase following adenovirus immunization, the level of mammalian target of rapamycin (mTOR) activity, the amount of costimulation and the duration of antigen availability act together to define the magnitude, phenotype, and functionality of memory CD8(+) T cells. Modulation of these factors can be used to selectively manipulate memory formation.