Symposium Archive

The effects of pathogen associated molecular patterns (PAMPs) circulating in the maternal bloodstream on the developing embryo are largely unknown. Cell wall peptidoglycan is a universal bacterial PAMP recognized by innate immune Toll-like receptor 2 (TLR2) resulting in inflammation. During meningitis, this interaction initiates widespread neuronal death with subsequent severe permanent sequelae. Recent evidence indicates cell wall crosses from the maternal circulation across the placenta into the developing fetal brain. In contrast to the postnatal case, the fetal brain escapes inflammation and neuronal death. Rather, neurons experience an induction of the transcription factor FoxG1 resulting in neuroproliferation. The proliferative response leads to a 50% greater density of neurons in the cortical plate at birth. Cell wall released by antibiotic treatment of maternal infection yields the same result. Neuroproliferation requires TLR2 and is recapitulated in vitro by TLR2/6, but not TLR2/1 ligands. The fetal neuroproliferative response is followed by abnormal cognitive behavior after birth. The bacterial cell wall-TLR2 signaling axis is a new participant and important modulator of microbial-host/embryo signaling.

During persistent antigen stimulation, CD8+ T cells show a gradual decrease in effector function, referred to as “exhaustion”, which impairs responses in the setting of tumors and infections. We have comprehensively analyzed changes in chromatin accessibility and gene expression in CD8+ T cells during acute and chronic viral infections in vivo and upon stimulation in vitro, and have defined a pattern of chromatin accessibility specific for T cell exhaustion, characterized by enrichment for consensus binding motifs for NFAT and Nr4a transcription factors. Anti-PD-L1 treatment of tumor-bearing mice resulted in cessation of tumor growth and increased granzyme and serpin expression by the tumor-infiltrating cells, with only limited changes in gene expression and chromatin accessibility. We have also developed a CAR T cell model that is effective against tumors bearing human CD19, and have shown that the chromatin accessibility pattern of CAR tumor-infiltrating T cells resembles that of endogenous tumor-infiltrating T cells as well as exhausted cells in the LCMV Clone 13 model of chronic viral infection. We expect that our studies will advance our molecular understanding of T-cell exhaustion in cancer and other inflammatory settings.

Streptococcus pneumoniae (Pneumococcus), a major respiratory bacterial pathogen is associated with significant morbidity and mortality in both the US and globally. Capsule based vaccines are available for pneumococcal infections, but their efficacy is limited. Additionally, the evolution of non-vaccine serotypes poses an ongoing challenge to the efficacy and sustenance of existing vaccines. Pneumococcal infections are usually precipitated by some risk factor (usually an inflammatory event) that allows the transition of a commensal bacterial organism into an active pathogen, causing infections of varying severity from sinusitis to pneumonia and sepsis. Respiratory co-infection by Influenza A Virus (IAV) is one of the most significant risk factor for the commencement of pneumococcal infections. IAV infection overwhelmingly recruits inflammatory monocytes and neutrophils, which leads to significant tissue damage and creates a breeding ground for pneumococcal and/or other airway bacterial infections. The early events of IAV infection are sensed by a complex network of innate lymphoid and myeloid cells that promote damaging inflammation in the NP and lung.
Using mono pneumococcal and pneumococcus-IAV mouse co-infection models, we found the conditionally protective role of interleukin 17A (IL-17A). While IL-17A promoted inflammation to overcome the NP immune tolerogenic threshold that eventually resulted into the resolution of asymptomatic NP pneumococcal colonization, we observed a paradoxical role of IL-17A as being a contributor to immune pathology during a pneumococcal-IAV severe co-pathogenesis. Therefore, the role of IL-17A requires to be further delineated as to whether respiratory bacterial infections might better be controlled by suppressing tissue inflammation rather than promoting it, especially in a setting of an inflammatory risk factor. Further studies in our lab are underway to characterize tissue specific immune cell phenotypes (NP, Lung) and regulation of IL-17A effector response.

Lyme Disease (LD) is a vector-borne bacterial infection caused by Borrelia burgdorferi (B. burgdorferi). If not diagnosed and treated promptly, the infection can disseminate into the central nervous system (CNS), resulting in neuroimmune changes and neuroborreliosis. Although it is known that neuroborreliosis can affect cognition and psychological state in humans, a mouse model that replicates some or all of these changes in behavior has not previously been established. In our new mouse model of neuroborreliosis, we demonstrated that intradermal infection with 106 spirochetes results in dissemination of B. burgdorferi into the brain, dura, and deep cervical lymph nodes of mice, as early as 5 days post infection (dpi), and persisted in tissues as long as 45 dpi. At both time-points we observed neuroimmune changes in B cell and T cell subsets within CNS tissues, and these changes paralleled the neuroimmune changes that have been reported in patients. We performed behavioral tests to evaluate changes in psychological state and neurocognitive function in mice that had been infected for 45 days. We observed no changes in anxiety or depression. However, infected mice demonstrated significant deficits in short-term learning and memory, consistent with the neurocognitive deficits that patients with neuroborreliosis can experience. Collectively, our data suggest that our mouse model of neuroborreliosis may be appropriate for understanding the mechanisms that underlie neurocognitive deficits related to neuroborreliosis and associated neuroimmune responses.

Type 3 secretion system needle proteins are PAMPS that are reported to interact with NAIP/NLRC4, TLR2, and TLR4 to activate immune responses. In this study we analyzed the T3S needle protein (BscF) from Bordetella pertussis for its ability to induce immune signaling and to act as an immunogen or an adjuvant, either alone or in the context of an acellular pertussis vaccine. BscF was found to interact with TLRs 2 and 4 like other T3S needle proteins to activate NF-κB/AP-1 signaling, with a preference for TLR4 in mice. Additionally, BscF was found to utilize NLRP3 and caspase 1 to induce IL-1β that could be blocked by cytochalasin D, suggesting involvement of endocytosis in needle protein induced signaling. Using a combination of pharmacological-inhibition and siRNA-inhibition of clathrin mediated endocytosis we demonstrated that blockage of endocytosis decreased BscF and YscF induced NF-κB/AP-1 signaling. Further, we demonstrate that CD-14 and MD-2 are involved in the the TLR-mediated signaling by T3S needle proteins. Finally, we show that BscF is a strong immunogen but does not induce protective immunity against B. pertussis when used alone. However, when BscF is included in a laboratory created acellular pertussis vaccine it promotes increased protection, possibly by increasing Th1 and Th17 responses relative to vaccine formulations lacking BscF.

Sepsis is a complex immune disorder that is characterized by systemic hyperinflammation, and has a mortality rate of 20-50% (1). With no effective treatment at hand, sepsis resulting from injury and infections associated with postoperative care is a major healthcare concern. Recent studies, including several from our laboratory, implicate aberrant neutrophil function as one of the underpinnings for hyperinflammation characteristic of sepsis. A recently established paradigm of neutrophil activation is formation of neutrophil extracellular traps (NETs) which are decondensed chromatin fibrils coated with granular proteases and histones. Overproduction of NETs has been shown in several inflammatory diseases including sepsis, acute lung inflammation, myocardial infarction, vasculitis, rheumatoid arthritis, lupus, atherosclerosis, and Alzheimer’s. Inhibition of exuberant NET formation thus can improve disease outcome in sepsis as well as other NET associated inflammatory diseases. In this regard, we have identified a novel anti-inflammatory biomolecule that inhibits neutrophil NET formation and mitigates experimental sepsis. Importantly, this biomolecule (termed IHBM) does not affect the ability of neutrophils to produce reactive oxygen species (ROS), an important defense mechanism of neutrophils. Furthermore, IHBM treatment potentiates resolution of inflammation by modulating macrophage phenotype. This is of importance because neutrophil NETs in many inflammatory diseases have been shown to prime macrophages to produce inflammatory mediators, thus promoting inflammation. Overall, these exciting findings posit IHBM as an extremely attractive prophylactic and therapeutic agent. We are currently examining the mechanistic underpinnings of IHBM-mediated inhibition of NET formation and its therapeutic potential in various disease conditions with exuberant NET formation.

Lyme disease (LD) is the most prevalent vector-borne disease in North America1. Clinical manifestations can include arthritis, carditis, and neurological complications such as meningitis, cranial and peripheral neuritis, facial nerve palsy, encephalitis, and cognitive decline2. This multisystem disease is caused by infection with a genetically heterogeneous group of spirochetes, including Borrelia burgdorferi (Bb) and related species. Multiple inter- and intra-isolate differences in colony morphology, adaptive gene expression, and infection phenotype have been noted that are not easily explained by differences in genetic content 8 , suggesting alternative mechanisms of phenotypic heterogeneity (i.e. epigenetic gene regulation). DNA methylation is the product of methyltransferase enzymes (MTases), and has been described in both prokaryotes and eukaryotic organisms as a mechanism of epigenetic gene regulation. Two distinct m6A MTases have been described in Bb as part of restriction modification (RM) systems which play a pivotal role in defense against foreign DNA, and evidence exists for the presence of yet unidentified MTases. The specific recognition and/or methylation sequence motifs have not been identified for any Bb MTase, and it is currently unknown whether the MTases associated with these RM systems can also function as gene expression regulators.

We and others have recently noted phenotypic differences such as neurotropism and in vitro virulence factor expression between a Bb tick isolate (Bb_B31) and a human neuroborreliosis clinical isolate (Bb_297). Interestingly, the complement of identified MTases differs between these isolates. Epigenetic regulation of gene expression may have effects on host range, tissue tropism, and the ability to induce disease pathology. As such, a more comprehensive characterization of the DNA methylation systems in Bb and their effects on gene regulation is crucial for a thorough understanding of Bb virulence and the pathobiology of Lyme disease.

Our central hypothesis is that the global DNA methylation patterns are distinct between Bb isolates. Here, we propose to use single-molecule real time (SMRT) sequencing to determine specific methylated DNA bases, evaluate the contributions of specific MTases to these modifications, and to compare the methylation pattern between Bb_B31 and Bb_297. The use of SMRT sequencing to directly evaluate specific DNA modifications is an emerging technology that is expected to have increasing impacts on epigenetics research as the technology advances. Next, we will use whole transcriptome RNA-seq to determine the effects of specific methylation profiles on gene expression, and evaluate differences between strains to identify potential candidates relevant for Bb virulence that could be exploited for the development of novel prophylactic or therapeutic treatments for Lyme disease.

Macrophage plasticity is essential for innate immunity and tissue remodeling, but signaling mechanism(s) regulating their functional phenotypes remains to be defined in depth. Here we report that IFNg priming of naïve macrophage induces store-mediated Ca2+ entry, and inhibition of this Ca2+ entry attenuates production of M1- inflammatory mediators. Functional analysis revealed that both in in-vitro and in-vivo conditions, ORAI1 channel function as a primary contributor to the basal Ca2+ influx in macrophage. Whereas IFNg-induced Ca2+ influx into macrophage was mediated by TRPC1. Deficiency of TRPC1, displayed an abrogated IFNg-induced M1- associated immune mediators in macrophages in vitro and in vivo. Notably, in a preclinical model of peritonitis due to Klebsiella pneumoniae (KPn) infection, macrophages showed increased Ca2+ influx, which was again TRPC1-dependent. Furthermore, macrophages from KPn infected TRPC1-/- mice showed inhibited expression of M1-associated signature molecules. Overall, these results suggest that TRPC1 mediated Ca2+ influx is induced and shapes MФ polarization to M1-pro-inflammatory functional phenotype.

Toll-like receptors (TLR) are a family of innate receptors that can be found on the cell membrane or intracellularly and are important in the first line of defense against bacterial and viral pathogens. We have shown that purified type III secretion (T3S) system needle proteins from a number of Gram-negative bacteria are potent TLR2 and TLR4 agonists; inducing NF-B/AP-1 signaling and robust inflammatory cytokine release. TLR2 and TLR4 have been shown to signal from the plasma membrane and endosomal compartments. In the current study, we investigated the role that endosomal signaling plays in T3S system needle protein induced NF-B/AP-1 signaling. To determine NF-B/AP-1 activity, we used human embryonic kidney 293 HEK-Blue^TM cells that express the reporter gene SEAP under NF-B/AP-1 control. In addition, we used human THP-1 cells, a human monocytic cell line. Pharmacological inhibition and siRNA knockdown were used to specially target various stages of clathrin-mediated endocytosis prior to T3S system needle protein stimulation in both HEK-Blue^TM and THP-1 cells. In addition, blocking antibodies were used targeting CD14 and MD2 in HEK-Blue cells. NF-B/AP-1 stimulation was assessed by measuring SEAP in detection medium. Culture supernatants were collected from THP-1 stimulated cells and TNF-was determined by ELISA. T3S system needle proteins activated NF-B/AP-1 signaling from endosomal compartments in HEK-Blue^TM cells. Interestingly, needle protein induced NF-B/AP-1 activation was controlled by a clathrin/dynamin dependent endocytosis mechanism, as clathrin-dependent endocytosis inhibition significantly reduced NF-B/AP-1 signaling in HEK-Blue^TM cells. This was further confirmed by demonstrating that TNF- production from THP-1 cells was significantly reduced after exposure to endocytosis/dynamin inhibitors. These observations further elucidate the innate signaling pathways activated by T3S system needle proteins.

Hypersensitivity to food is often reported to trigger or exacerbate neuropsychiatric symptoms such as anxiety and autism. Although a growing amount of studies support this observation, how peripheral allergic responses lead to aberrant behavior remains elusive. Because mast cells are major responders in type I hypersensitivity and capable of migrating to the brain, they may play a role in communicating peripheral inflammatory events to the central nervous system, likely via the release of histamine, cytokines, and other inflammatory factors which adversely affect brain function. Using a mouse model of milk allergy, we tested our hypothesis that food-allergen sensitization would elicit behavioral changes and that the number of their brain mast cells would be elevated. Four-week-old (young) and 10-month-old (old) male and female C57BL/6 mice were orally sensitized to whey proteins (WP) for 5-weeks, followed by an oral WP challenge. Behavioral analysis revealed that WP-sensitized males showed significantly less digging activity than sham males in both age groups, while no apparent difference was observed in females. Brain mast cells were predominantly localized between the lateral midbrain and medial hippocampus, and increased in WP-sensitized young, but not old, male brains. Perivascular astrocyte hypertrophy was also observed in the old WP-sensitized mice, although this morphological difference was not apparent in the younger mice. Our results demonstrated that our milk allergy mouse model exhibits behavioral abnormality and changes in the number of brain mast cells, suggesting a potential role of mast cells in food allergen-induced behavioral dysfunction.

Enhancers are regulatory regions of the genome that can control gene expression independent of their distance from the transcription start site (Heintz et. al, 2015). Enhancer may exist in inactive, primed, or active states (Levine et al, 2014) that in turn can determine the fate of the gene expression with which the enhancer is associated. Enhancer functions have been found to be widely dysregulated in diseases such as cancer. However, how these enhancers change especially in response to stimulus such as inflammation needs to be further elucidated. Our study is to investigate the mechanism of enhancer action in regulating gene expression in lung cancer cells. To study this phenomenon, we challenged A549 lung adenocarcinoma cells with proinflammatory cytokine IL-1β for different time points (0mins, 30mins, 60mins, 120mins, and 5 hours) and observed changes in gene expression. To identify spontaneous activation of enhancers across the genome in response to proinflammatory stimulus, we analyzed nascent RNA transcription by precision run-on sequencing (PRO-seq) (Mahat et al, 2016). We found nascent RNA transcription at various locations of genome in response to IL-1β. We are now bioinformatically identifying the generation of novel enhancers as well as activation of already identified enhancers genomewide, in response to IL-1β. We are currently performing RNA-sequencing experiments to determine timing of enhancer activation, and if enhancer is activated prior to gene activation. Our future direction is to identify a mechanism of how these enhancer RNAs regulate expression of their corresponding genes.

Pseudomonas aeruginosa is a key human nosocomial pathogen, responsible for ∼10% of hospital acquired infections and is frequently associated with adverse medical outcomes. Mitophagy has recently been identified as an effective way to control the intracellular P. aeruginosa in mammalian cells. In the present study, we demonstrate a novel role of miR302-367 cluster in regulating the mitophagy-mediated antibacterial response. The results showed that miR302-367 cluster expression was significantly enhanced after P. aeruginosa infection. Forced expression of miR302-367 cluster accelerated the mitophagic response in macrophages, thus promoting the induction of reactive oxygen species (ROS) and decreasing the survival rate of intracellular P. aeruginosa. On the other side, transfecting with miR-302s inhibitors increased P. aeruginosa survival. Nevertheless, macrophage-mediated P. aeruginosa phagocytosis was not altered after miR-302s overexpression or inhibition. Blocking autophagy with the inhibitor 3-methyladenine (3-MA) or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-302s to promote mitophagy and P. aeruginosa elimination. Furthermore, our study demonstrated that miR302-367 cluster bound to the 3’-untranslated region of nuclear factor kappa B (NF-κB), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular P. aeruginosa by suppressing NF-κB expression. Taken together, our results elucidate that miR302-367 cluster could function as the new regulator in mitophagy-mediated P. aeruginosa elimination by manipulating ROS/NF-κB circuits, and pinpoint an unexpected functional link between miRNA and mitophagy.

This work is supported by NIH grant: R01 AI109317 and P20 GM113123.

Apolipoprotein E (ApoE) is the major carrier protein that mediates, via activating ApoE receptors, the transport and delivery of cholesterol and other lipids in brain. Three different genetic isoforms of ApoE (ApoE2, ApoE3, ApoE4) exist in humans and their relative expression levels impact HIV-1 infection, HIV-1/AIDS disease progression, and cognitive decline associated with HIV-1 associated neurocognitive disorder. Because HIV-1 Tat, a viral protein essential for HIV-1 replication, can bind to low-density lipoprotein receptor-related protein 1 (LRP1) that controls ApoE-cholesterol uptake in brain, we determined the extent to which different isoforms of ApoE affected Tat-mediated HIV-1 LTR transactivation. Using U87MG glioblastoma cells expressing LTR-driven luciferase we found that LRP1 as well as ApoE2, ApoE3 and ApoE4 all affected Tat-mediated HIV-1 LTR transactivation. A specific LRP1 antagonist, receptor associated protein, and siRNA knockdown of LRP1 both restricted significantly Tat-mediated LTR transactivation. Of the three ApoEs, ApoE4 was the least potent and effective at preventing HIV-1 Tat internalization and at decreasing Tat-mediated HIV-1 LTR transactivation. Further, Tat-mediated LTR transactivation was attenuated by an ApoE mimetic peptide, and ApoE4-induced restriction of Tat-mediated LTR transactivation was potentiated by an ApoE4 structure modulator that changes ApoE4 into an ApoE3-like phenotype. These findings help explain observed differential effects of ApoEs on HIV-1 infectivity and the prevalence of HAND in people living with HIV-1 infection and suggest that ApoE mimetic peptides might be used as a therapeutic strategy against HIV-1 infection and associated neurocognitive disorders.

Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to cleave foreign DNA to defend against invading nucleic acids. Recently, although huge interest has been aroused in CRISPR-Cas9 due to its gene editing potential, little is known about how bacteria self-regulate the CRISPR-Cas immune response. Here, we show that Pseudomonas aeruginosa virulence factor CdpR ( ClpAP -degradation and pathogenicity regulator) acts as a negative regulator of CRISPR-Cas systems to inhibit the process of immunization and immunity. Furthermore, we find that CdpR represses quorum sensing regulators, especially LasI/RhlI axis, to conditionally modulate CRISPR-Cas system functionality. Meanwhile this process needs the virulence factor regulator ( Vfr ) binding to cis-response elements in cas1 promoter to regulate cas operon activities. Importantly, CdpR influencing Vfr represses cas genes expression and impairing immunization and immune-memory of CRISPR-Cas against phage infection and self-targeting. Mechanistically, CdpR disrupts CRISPR-mediated cleavage of specific bacterial endogenous sequences by repressing CRISPR-Cas activity. These results indicate that CdpR suppresses CRISPR-Cas systems to prohibit development of autoimmunity but heightening infection risk by phages. Collectively, our findings have elucidated a mechanism by which CRISPR-Cas systems may subject intense negative regulation to keep homeostasis, while efficiently counteract bacteriophage ruthless invasion.

This work is supported by NIH grant: R01 AI109317 and P20 GM113123.

Host HLA class-II (HLA-II) allelic polymorphisms modulate susceptibility to severe Streptococcal toxic shock syndrome (STSS), caused by Group A Streptococcus bacteria (GAS), through their ability to regulate T cell responses to GAS superantigens (SAg), pivotal mediators of STSS. FoxP3 expressing CD4+ regulatory T cells (Tregs) play an important role in the regulation of immune responses and suppress excessive inflammatory responses that are detrimental to the host. Although a number of markers including the widely used CD25 and FoxP3 exist for the identification of Tregs, these markers do not distinguish between activated Tregs and bona fide Tregs that have suppressive function. However, neither the induction of functionally suppressive Tregs nor the markers that help their identification have been studied in STSS. Specifically, the possible role of latent Transforming growth factor-beta (TGF-β), Latency Associated Peptide (LAP), and its membrane anchor Glycoprotein A Repetitions Predominant (GARP or LRRC32) expressing Tregs that mediate suppression of inflammatory responses elicited by activated effector cells is unknown. To gain such insight, we performed phenotypic analysis of activated Tregs expressing LAP, GARP and FoxP3 in response to SAg-Streptococcal Mitogenic Exotoxin Z (SmeZ) stimulation of splenocytes from transgenic mice expressing HLA-II alleles associated with either protection (DR15) or neutral risk (DR4/DQ8) for STSS. When compared to DR4/DQ8, SmeZ presentation by the protective DR15 allele significantly promoted the induction of both FoxP3+Tregs (p=0.01) and increased frequency of GARP+LAP+ and GARP+LAP- activated Tregs. Conversely, SmeZ presentation by DR4/DQ8 promoted significant induction of FoxP3 lowGARP-LAP+ activated Tregs (p=0.04) that correlated with high frequency of CD4+CD25+, SmeZ-reactive TCRVß11+CD4+CD25+T cells and significantly increased IL-2/IFN-γ producing CD4+CD25+ cells (p=0.03, 0.01 respectively). The differential induction of FoxP3+/lowGARP+/-LAP+/- activated Tregs potentiated by HLA-II alleles associated with either protection or neutral risk for STSS in vitro to SAgs and during in vivo GAS infections provides new understanding of suppressor/effector mechanisms underlying disease outcomes in STSS.

Zika virus is an emerging pathogenic arbovirus of humans in the western hemisphere. Zika infection results in clinical manifestations ranging from mild symptoms including fever, rash, joint and muscle pain, and conjunctivitis, to more severe symptoms in the CNS including Guillain-Barré syndrome. Zika infection has also been shown to cause microcephaly and other fetal brain defects during pregnancy. With Zika’s potential spread into new geographical areas, it is important to define the vector competence of native mosquito species. We tested the vector competency of host-seeking mosquitoes, primarily Aedes (Ae.) vexans, collected from northwestern Minnesota and northeastern North Dakota. Colonized Ae. aegypti (Costa Rica strain) were used as a positive control vector for comparison. Mosquitoes were fed blood containing Zika virus and two weeks later were tested for viral infection and dissemination. Aedes vexans were susceptible to midgut infection (28% infection rate, n=60) but displayed a modest midgut escape barrier (3% dissemination rate, n=60). Co-fed Ae. aegypti displayed significantly higher rates of midgut infection (61%, n=22) and dissemination (22%, n=22). To test for potential salivary gland barriers to virus transmission, mosquitoes were inoculated with virus and 16-17 days later, tested for their ability to transmit virus. Unexpectedly, the transmission rate was significantly higher for Ae. vexans (34%, n=47) than that for Ae. aegypti (5%, n=22). Thus, Ae. vexans populations from the northern Great Plains can serve as potential vectors for Zika virus should the virus be introduced into the region.

The innate immune system is an important first line of defense against a pathological challenge and the sympathetic nervous system is an important regulatory mechanism for the innate immune response. An increased recurrence of the contagious respiratory disease, pertussis, to levels not seen since the early 2000’s can be attributed to the use of an attenuated acellular vaccine against Bordetella pertussis. However, it is not known whether this pertussis re-emergence is a result of decreased protection or differences in the immune response stimulated by the acellular vaccine when compared to the natural infection. Early investigations also demonstrated a protective effect in response to sympathetic nervous system activation using a pertussis animal model. Therefore, a broad, long-term objective of this investigation is to provide a better understanding for the nature and significance of the host response to B. pertussis infection. This information would be fundamental in clarifying the mechanisms of pertussis recurrence in the United States. The goal of this proposal is to increase our comprehension of inflammatory signaling molecules generated by immune cells exposed to B. pertussis and the modulatory effects of the sympathetic nervous system on this biochemical profile. The North Dakota Flow Cytometry and Cell Sorting core facility will be employed to achieve the stated goal by utilizing a robust approach to identify and quantitate amounts of these inflammatory molecules. Using the bacterial endotoxin, lipopolysaccharide as a control of inflammatory initiation, chemokine and cytokine profiles will be generated from human monocytes challenged with B. pertussis needle protein in the absence or presence of selective adrenergic receptor ligands. Preliminary data, using a fluorescent multiplexed bead-based immunoassay, characterizes modified chemokine and cytokine profiles generated from human monocytes challenged with B. pertussis needle protein and concurrently incubated with or without epinephrine. These initial studies will be expanded by identifying the type and functional consequences of selective adrenergic receptor activation that alters B. pertussis inflammatory signaling. Results from this enhanced experimental approach will provide a detailed summary of the host inflammatory response to a B. pertussis challenge as well as identifying the adrenergic receptor type responsible for modulating these chemokine and cytokine profiles. By expanding the breadth and depth of knowledge for this recurring infectious disease, newer, safer and more efficacious treatments can be developed in response to this emerging public health threat.

Multidrug resistance bacteria (MDR) with increasing prevalence of biofilm-associated infections present alarming challenges, which demands innovative therapeutic strategies to control bacterial infection. As a representative of the multi-drug resistant pathogenic bacteria, Pseudomonas aeruginosa has evolved into a head-scratching adversary that causes stubbornly high morbidity and mortality especially in nosocomial infection. Here we provide a novel guanidinium-functionalized polycarbonate (DP10) to deal with this problem. We have designed a novel class of polymers, guanidinium-functionalized polycarbonates (e.g., DP10) and demonstrated high efficacy of antibacterial activities to P. aeruginosa, A. baumannii , E. coli and K. pneumaniae , indicating DP10 has the great potential to counteract serious infections with multiple MDR bacteria. Here, we investigated the mechanism by which DP10 inhibits the P. aeruginosa and reveal that DP10 functions at prohibiting biofilm formation and quorum sensing signaling as well as blocking the release of virulence factors. Therefore, our findings suggest that DP10 may serve as a viable means for treating P. aeruginosa and other MDR bacterial infections. This work is supported by NIH grant: R01 AI109317 and P20 GM113123.

Alternatively activated macrophages (M2 MФ) function to dampen host inflammatory responses and promote wound healing and tissue repair. As the blood brain barrier (BBB) restricts the flow of both cells into the brain, understanding how M2 MФ extravagate into the central nervous system (CNS) is of great interest. In a mouse model of neurocysticercosis (NCC) we previously demonstrated that M2 MФ are essential in containing neuropathology in helminth infected CNS. Analysis of helminth infected mice revealed an abundant expression of galectin-7 that recognize galactose moieties in brain endothelial cells. The Galectin-7-/- NCC mice displayed a reduced accumulation of M2 MФ in parasite infected brain. This reduction in M2 MФ numbers in the CNS was correlated with an increased neuropathology and shortened survival. The use of adoptive transfer of fluorescent labeled cells demonstrated a decreased influx of M2 MФ into the CNS of helminth infected NCC brains. The use of in-vitro transmigration assay conclusively demonstrated a key function of Galectin-7 expressed on endothelial cells in centrally regulating M2 MФ entry into the CNS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is fatal within 3-5 years of diagnosis. Most cases have environmental causes, but environmental risk factors have not been identified. We reported that mortality rates for motor neuron disease (MND) are associated with the use of well water. Because users of well water have higher risks of waterborne disease than users of municipal water, we hypothesized that MND might be caused by a waterborne pathogen(s). Since several waterborne pathogens cause pneumonia, we examined the association between pneumonia and a later diagnosis (dx) of MND/ALS. We used a medical record data-base with data on ~ 69 million individuals, MarketScan™ 100% Sample claims data. MarketScan™ is a database of employer-based health care claims that includes inpatient admissions in 10 geographically dispersed areas of the US. Exposure was classified as a principal dx of pneumonia (excluding “rule-out” diagnoses). Events were a principal dx of MND and/or ALS. We defined baseline (t0) as the date of pneumonia dx for exposed individuals and the beginning of data (enrollment) for unexposed individuals. Event time (t1) was the dx date with MND/ALS for events or the date of censoring for non-events. Follow-up was the interval between t0 and t1. We used multivariate regression and Cox proportional hazards.

We excluded individuals with prevalent disease. The sample included 782 MND events among 68,689,301 persons and a random sample of 1,000,000 non-events. Among persons with pneumonia, 32 cases of MND were diagnosed for a crude incidence rate of 211.1 cases/100,000 person years. Among persons without pneumonia, there were 750 cases of MND for a crude incidence rate of 11.4 cases/100,000 person-years. The median time between the pneumonia dx and MND dxs was 233 days (mean 314 days; range 5 – 1,508). The crude relative-hazard for motor-neuron disease given past-pneumonia was 14.31 (95% C.I. 10.05 – 20.04). The final age-adjusted relative hazard of MND given a past diagnosis of pneumonia was 3.01 (95% C.I. 2.09 – 4.34, P > 10^-6). Our findings implicate pneumonia, and/or the causal pathogen(s), and/or its treatment in the etiology of ALS. Bacterial pneumonogens are a biologically plausible cause ALS. These agents cause neuroinflammation (e.g., via lipopolysaccharide [LPS]) and neurologic disease in animals and in humans. Our data suggest that bacterial pneumonogens should be explored as potential animal models of ALS.

Through clinical studies in humans, allergies have been demonstrated to be comorbid with neuropsychiatric disorders. Induction of circulating proinflammatory cytokines, IgE-mediated mast cell degranulation, impaired intestinal absorption or altered metabolism of neurotransmitter precursors, are potential peripheral events that may lead to modifications in brain function. We hypothesized that these events would induce transcriptomic and epigenomic changes in the brain due to food-allergy-mediated gut inflammation. We used a mouse model of cow’s milk allergy to profile these changes. Male and female 4-week-old C57BL/6J were orally sensitized to a milk protein, β-Lactoglobulin (BLG) in the presence of cholera toxin as an adjuvant over a 5-week period. At week 6, the mice were challenged with a higher dose of BLG. A set of 10 male mice to be used for sequencing were sacrificed one day later to harvest the brain, ileum, and serum. The remaining mice were evaluated behaviorally post-challenge and were challenged a second time prior to sacrifice and collection of brain, ileum, and serum. Concentration of BLG-specific IgE-specific was quantified using ELISA. The brain was further subdivided into 4 regions and nuclei acids were isolated for transcriptomic and epigenomic analyses. BLG-specific IgE was increased in the sensitized mice compared to sham-treated control mice, validating their sensitivity to the milk protein. RNA-sequencing showed small but significant transcriptional changes in a set of genes expressed in the brain regions, including various long noncoding RNAs such as Pisd-ps1, Miat, and Rbm3. Transcriptional changes occur in the brain after food-allergy-induced peripheral inflammation. Additional studies to validate our RNA-sequencing results with RT-qPCR are warranted.

The mucosal lining of the intestine faces many insults and so it must be able to heal itself. This complex process of intestinal mucosal healing is influenced by physical forces of peristalsis, villous motility, interactions with luminal contents, and intraluminal pressure. Specifically, increased intraluminal pressure is present in diverse intestinal pathologic states such as, postoperative ileus, complete bowel obstruction, irritable bowel syndrome, and chronic inflammatory states of Crohn’s disease and ulcerative colitis. In a model of jejunal, partially obstructed, Roux-en-Y anastomosis, there is reduced peristalsis and increased intraluminal pressure that slows ulcer healing. The influence of such physical forces on intestinal epithelial barrier function, the gut microbiome, and the immune system are still poorly understood with respects to intestinal mucosal healing. We will identify what regulatory bacteria, immune and/or intestinal cells have been effected by increased intraluminal pressure and how this leads to the invasion of adverse immune secretions and potentially pathogenic commensals inhibiting mucosal healing in the Roux-en-Y anastomosis limbs. We hypothesize that increased intraluminal pressure and reduced peristalsis of Roux-en-Y anastomosis causes a commensal bacteria imbalance that impedes proper toll-like receptor signaling crosstalk with the immune milieu and intestinal epithelial cells and subsequently leads to inadequate induction of the mucosal healing pathways. Upon successful completion of this project, we will have further defined mechanisms involved in mucosal healing. Delineating the commensal flora switch to a potential pathogenic intestinal environment and how this may impact or coincide with the immunological response, will enable us to drive future developments of restorative therapeutics for mucosal injury. These restorative therapeutics would include targeted probiotic therapy, targeted induction of immune signaling pathways, and adoptive transfers of regulatory immune cells. Lastly, these types of therapeutics could impact the treatments of minor ailments of stress ulcers, postoperative ileus, and bowel obstruction, to major diseases of Crohn’s disease, ulcerative colitis, and cancer.

Inducing sustained, robust CD8+ T cell responses is necessary for therapeutic intervention in chronic infectious diseases and cancer. Unfortunately, most adjuvant formulations fail to induce substantial cellular immunity in humans. Attenuated acute infectious agents induce strong CD8+ T cell immunity, and are thought to therefore represent a good road map for guiding the development of subunit vaccines capable of inducing the same. However, recent evidence suggests that this assumption may need reconsideration. Our data demonstrate that the rules determining the T cell responses to vaccination differ substantially from those induced by infectious challenge. As such, we would argue that research in pursuit of cellular immunity-inducing vaccine adjuvants should no longer follow only the infection paradigm.

Syphilis, caused by the spirochete Treponema pallidum, continues to be a prominent disease in low- and middle-income countries and is re-emerging as a significant public health threat in high-income countries. Syphilis elimination will require development of an effective vaccine, yet a promising vaccine candidate has thus far remained elusive. One of the key mechanisms of pathogenesis of T. pallidum is the capacity of the pathogen to disseminate rapidly and widely within the host, invading every organ and tissue and causing the varied and serious sequelae associated with syphilis. We have previously identified a dual functioning T. pallidum adhesin/protease, designated Tp0751, that attaches to the host vasculature, degrades host components and aids in entry of T. pallidum to the bloodstream and treponemal dissemination via the vasculature. Immunization with Tp0751 provides sterile protection against dissemination in the syphilis animal model upon challenge with virulent T. pallidum, as evidenced by the lack of induction of a productive infection upon transfer of tissue from immunized animals to naïve animals, in comparison to induction of an active infection upon tissue transfer from unimmunized, challenged animals. Interestingly, Tp0751 is structurally similar to a Neisseria meningitidis lipocalin protein that is common to two licensed meningococcus vaccines. The shared lipocalin scaffold may contribute to the widespread dissemination capacity of these highly invasive pathogens. Collectively these findings provide convincing evidence that Tp0751 is a promising and effective syphilis vaccine candidate and may reveal a novel mechanism for tissue invasion by disseminating pathogens.

Type III secretion systems (T3SS) are nanomachines used by some Gram-negative bacteria to communicate directly with targeted eukaryotic cells. A typical T3SS is comprised of a basal structure (a syringe) that spans the entire bacterial envelope and serves as the anchor for an external needle that possesses a tip complex (TC). The TC recognizes host cell surfaces and, upon sensing contact, inserts the proteins into the host membrane to create the translocon pore. It is through the translocon that effector proteins are injected to alter normal cellular functions. The Shigella flexneri T3SS senses contact with human intestinal cells to inject effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). Our group was the first to identify the components of the Shigella TC and to work out the events that occur within the TC that lead to induced secretion. That work has evolved into an ongoing investigation of the biochemistry, structure and function of the translocon pore component invasion plasmid antigen B (IpaB) which is anchored to the T3SS needle tip by IpaD. In parallel, we are exploring the structure and function of a large cytoplasmic complex that we propose forms the sorting platform at the base of the envelope-spanning basal body. The sorting platform is essential for effector protein selection and needle assembly, but it remains largely uncharacterized. We are working with other groups to use high throughput cryo-electron tomography to visualize the T3SS of Shigella flexneri in situ so that we can develop mechanistic models of Shigella type III secretion function and activation.

Lyme disease is the most prevalent tick-borne disease in the US with an estimated 300,000 cases per year. The causative agent of the disease is Borrelia burgdorferi (Bb) and is transmitted to humans by the bite of infected ticks. Bb causes a multi-system disorder including neurological complications (neuroborreliosis). Despite appropriate antibiotic treatment, up to 20% of all patients treated for Lyme disease experience Post-Treatment Lyme Disease Syndrome (PTLDS) characterized by fatigue, musculoskeletal pain, and neurocognitive complaints. The total direct medical costs of Lyme disease and PTLDS are estimated to approach $1.3 billion each year. Recently, our laboratory has demonstrated that 1) Bb can be detected in the brains of infected rats after IV infection; 2) Bb can cause neuroinflammatory changes in the brain that recapitulate those seen in human patients and nonhuman primate models; 3) Bb DNA and antigen persist after antibiotic treatment; and 4) antibiotic-killed Bb can induce an inflammatory response in the brain in vivo, and in glia cells in culture, suggesting that dead microorganisms and spirochetal debris that remain after antibiotic treatment can contribute to a sustained Bb-induced neuroinflammatory response in the absence of live microorganisms. We hypothesize that the inflammation and lingering symptoms of neuroborreliosis are due to the persistence of bacterial debris following antibiotic treatment.

Inflammatory disorders can be triggered by overactive immune responses directed against host/self tissues. Indeed, neuroinflammation-associated disorders such as multiple sclerosis and stroke can be triggered and/or exacerbated by overactive immune responses against nervous tissues. It is now evident that not only cognitive disorders, but classic mental diseases such as schizophrenia and depression, are related to neuroinflammation. It is thus remarkable that infection with parasitic helminths (worms) suppresses inflammation in a variety of immune disorders. Ongoing clinical trials using worm egg in patients with multiple sclerosis are showing promising results. Hence, an understanding of helminth immunoregulatory mechanisms is likely to have enormous impact on novel strategies against inflammatory disorders. In this light, the regulation of inflammatory responses is mainly mediated via the innate immune system through myeloid antigen-presenting cells (APC) such as macrophages (MΦ) and microglia (MG). The functional phenotypes of MG and MΦ vary in response to external stimuli these cells receive through a wide variety of surface receptors. It is thought that worms or their products induce alternatively activated macrophages (AAM) that dampen inflammation and promotes wound healing and tissue repair. In contrast to the classically activation phenotype, alternatively activated MΦ (AA-MΦ) and MG (AA-MG) have the capacity to dampen host inflammatory responses and promote wound healing and tissue repair in certain chronic neuroinflammation-related diseases, as we and others have documented. Our studies using a mouse model of neurocysticercosis (NCC) demonstrated that AAMs are essential in containing neuropathology and disease severity.

Additionally, consistent with human NCC, highly antigenic tegument galactose/ galactosamine and glucosamine containing glycan molecules of the parasite are released and taken up by host cells in the CNS environment. As lectin receptors (LRs) are the major receptors for recognition of glycan antigens, a study of their role in AA-MΦ and AA-MG functions in the CNS during NCC are important for understanding how NCC and neuropathological conditions can be clinically controlled. We hypothesize that glycan antigens released by parasites will lead to differential expression of specific host LRs which will play a critical role in the development and trafficking of AAM into the CNS, expression of effector molecules, and regulation of CNS immunopathology. In this presentation, I will discuss some of I our recent findings showing LRs are differentially expressed in the CNS during NCC. Importantly, targeted LRs such as Galectin-7 controls extravasation of AA-MΦ into the CNS, whereas Galectin-3 controls AA-MФ functions in efferocytosing of neutrophils. The presentation will also discuss the significance of targeted Galecitn-3 and Galectin-7 in regulating AA-MΦ mediated control of CNS immunopathogenesis in murine NCC. The results from these studies have an enormous impact on the understanding of the mechanisms that regulate AA-MΦ functions to suppress the host inflammatory response during helminthic infections. Moreover, our findings provide new directions and approaches to manipulate AA-MΦ function to suppress inflammation in a variety of inflammatory disorders, particularly in CNS inflammatory diseases.

Neutrophils are the first responders for combating microbial infections. The traditional antimicrobial program of neutrophils constitutes phagocytosis followed by production of noxious agents such as reactive oxygen species (ROS) to kill internalized microbes. In addition, a recently discovered paradigm in neutrophil antimicrobial program is the formation of extracellular traps (Neutrophil Extracellular Traps, NETs), which are DNA fibrils expelled by these cells that are decorated with granular contents such as various proteases. NETs have been reported to play protective roles in infectious diseases by trapping, neutralizing and killing extracellular microbes. On the flip side, the toxic cargo of neutrophils can cause bystander tissue pathology, and an exuberant NET formation has been linked to development of many inflammatory diseases or disorders by promoting hyper activated immune response. It is thus clear that neutrophil functions and their turn-over in a disease condition need to be tightly regulated and fine-tuned in a microenvironment dependent manner. To harness the beneficial outcome of neutrophil functions while avoiding their potentially harmful effects, a clear understanding of the molecular mechanism underlying their homeostatic turnover and specialized functions such as NET formation is essential. Our research strives to attain these goals in the setting of acute pneumonic infections as well as chronic lung inflammatory conditions, by studying the host- and pathogen-mediated regulation of efferocytosis (a process by which professional phagocytes clear apoptotic cells) and neutrophil NET formation.

Long noncoding RNAs (lncRNAs) modulate various biological processes; however, their function in host immunity response against infection has remained elusive. Here, we identify an intergenic lncRNA MEG3 (linc-MEG3) as a tissue specific regulator for pulmonary immunity during bacterial infection. Among the 10 transcripts of linc-MEG3, transcripts 1 and 4 are main regulators due to being the most downregulated in mouse lungs after bacterial infection. Overexpression of linc-MEG3-4 in mice led to intensified inflammatory response, severe lung injury, systemic infection dissemination, and ultimately, increased mortality. Alveolar epithelial cells and alveolar macrophages (AM) were major cell populations that are targeted by linc-MEG3-4. As its 3’ sequences are complementary to microRNA-138 (miR-138), linc-MEG3-4 competitively binds miR-138 and thus releases its target IL-1b mRNA, resulting in intensified inflammatory responses. Our findings characterize linc-MEG3 as a novel pulmonary inflammatory regulator of bacterial infection through a miR-138/IL-1b/NF-kB circuit.

Currently 36 million people worldwide are infected with HIV-1, and it is estimated that 50% of those HIV-1 infected individuals are having HIV-1 associated neurocognitive disorders. HIV-1 transactivator of transcription (Tat) is a viral protein that is essential for HIV-1 viral replication. Tat can be secreted from infected or transfected cells and taken up by bystander cells via receptor-mediated endocytosis. Upon receptor-mediated endocytosis, Tat enters endolysosomes, from which it can be released into the cytoplasm and enters the nucleus to facilitate HIV-1 viral replication. We postulate that changes in endolysosome pH following LRP-mediated endocytosis play a key role in Tat-mediated HIV-1 LTR transactivation. In U87MG astrocytoma cells expressing the LTR-driven luciferase, we determined the extent to which modulating endolysosome pH affects Tat-mediated HIV-1 LTR transactivation. Because LRP is the major receptor that mediate apoE-cholesterol uptake in the brain, we also determined the extent to which different isoforms of apoE affect Tat-mediated HIV-1 LTR transactivation. We demonstrated that endolysosome de-acidifying agents enhance Tat-mediated HIV-1 LTR transactivation; whereas endolysosome acidifying agents block Tat-mediated HIV-1 LTR transactivation. In addition, we found that apoE4 is less effective in preventing Tat-mediated HIV-1 LTR transactivation, comparing apoE2 and apoE3. Furthermore, we demonstrate that a small peptide that contains the apoE receptor-binding region attenuated the Tat-mediated HIV-1 LTR transactivation. Our findings suggest that endolysosome acidification and apoE mimetic peptide might be used as a therapeutic strategy against HIV-1 infection and associated neurocognitive disorders.

Incidence of pertussis, a severe respiratory disease caused by Bordetella pertussis, have been on the rise. This resurgence has been linked to antigenic divergence in circulating pertussis strains as well as waning or ineffective immunity induced by the current acellular pertussis (aP) vaccine. The current aP vaccine replaced a whole cell pertussis (wP) vaccine in the 1990s due to adverse events associated with the wP vaccine. Alum-absorbed aP vaccine has been shown to elicit a strong antibody response and considerable Th2 type CD4+ T cells. In contrast, wP vaccine promotes Th1/Th17 type cellular immunity and associated opsonizing antibodies presumably via its PAMPs. We have previously demonstrated that BscF, a B. pertussis needle protein, acts as a strong TLR agonist. Here we characterized the contribution of BscF to a laboratory prepared aP vaccine-induced response. aP + BscF resulted in statistically greater pertussis-specific antibody titers, compared to sham aP+ PBS. BscF enhanced long-term aP-specific immunity, compared to either the alum or sham controls, as measured by increased numbers of central memory T cells. Moreover, the addition of BscF resulted in skewing the pertussis-specific immunity toward Th1 and Th17 responses. Mice immunized with aP + BscF demonstrated significantly reduced bacterial burden in their lungs 5 dpi. These findings suggest that the addition of BscF induced a strong Th1 and Th17 anti-pertussis immunity, and could be a novel additional component in the next generation aP vaccine.

Lyme disease caused by infection with the bacterial pathogen Borrelia burgdorferi (Bb) is the most prevalent vector-borne disease in North America. Clinical manifestations can include arthritis carditis and neurological complications such as meningitis cranial and peripheral neuritis facial nerve palsy encephalitis and cognitive decline. Current evidence suggests that treatment with antibiotics is effective at resolving active Bb infection
however upwards of 20% of treated patients continue to suffer from neurocognitive complications termed post-treatment Lyme disease syndrome (PTLDS). Why do some patients continue to suffer clinical manifestations after antibiotic treatment? The amber hypothesis of infectious disease proposes that dead bacteria and their debris persist after active infection is resolved and the constant slow leakage of debris stimulates inflammatory pathogenic processes. As such we hypothesize that dead Bb and debris persist in the CNS after antibiotic treatment and contribute to an ongoing inflammatory response.
SUMMARY OF FINDINGS: Using a rat model of neuroborreliosis Bb DNA and antigen persist in the CNS even after antibiotic treatment. Direct intracerebroventricular (ICV) injection of both live and antibiotic-killed Bb resulted in altered mRNA expression of cytokines/chemokines including granulocyte chemoattractant macrophage colony stimulating factor as well as a number of factors involved in the recruitment or stimulation of T cells. Preliminary data suggest a change in microRNA expression profiles after ICV injection of both live and dead Bb. Altered CD11b and GFAP protein levels indicate activation of microglia (or infiltration of macrophages) and activation of astrocytes in the CNS in response to ICV injection of both live and dead Bb. In conclusion we provide evidence of lingering Bb DNA and antigens in the CNS after antibiotic treatment and show that dead Bb stimulate changes in mRNA miRNA and protein levels using a rat model of neuroborreliosis. These findings support the amber hypothesis as a mechanistic explanation for PTLDS.

Calcium influx via store operated calcium entry (SOCE) activates downstream signaling pathways and gene expression. Indeed, SOCE channels are thought play essential role for immunity to infection. However, despite the evidence that Ca2+ influx shape several effector functions of macrophages, the specific Ca2+ channel involved in shaping M1-inflammatory functional phenotype in macrophages is yet to be identified. In this study, we demonstrate that exposure of IFN-γ, which is a key (M1) pro-inflammatory phenotype inducer in macrophage, produced an increased SOCE in-vitro. Blocking this increased SOCE by 2-APB treatment inhibited IFN-γ –initiated signaling pathway activation and production of M1-inflammatory signature mediators. Electrophysiological recordings revealed a TRPC1-SOCE current in IFN-γ –activated cells. Co-IP experiments demonstrated an increased STIM1-TRPC1 complex formation on plasma membrane confirming TRPC1-SOCE in IFN-γ –activated M1 cells. Importantly, macrophages from mice deficient for TRPC1, or ex-vivo macrophage cells exposed to siRNA for TRPC1, failed to display IFN-γ- induced SOCE. Likewise, these TRPC1-deficient macrophages displayed inhibited production of M1-inflammatory mediators and signaling events. Furthermore, peritoneal macrophages in mice deficient for TRPC1 failed to display IFN-γ induced SOCE in vivo. TRPC1 deficient macrophages displayed an attenuated expression of surface maturation markers and inflammatory mediators in response to IFN-γ in-vivo. As M1-inflammatory phenotype is a hallmark of bacterial infections, the role TRPC1 channels in Ca2+influx and its role in inflammatory mediator production was analyzed in a murine model of peritonitis due to Klebsiella pneumoniae (Kpn) infection. Ex-vivo macrophages from Kpn infected WT mice showed increased SOCE consistent with TRPC1 channel activation that was minimally detected in infected TRPC1-/- mice. Moreover, macrophages in KPN infected WT mice showed abundant expression of M1 associated molecules such as CXCL10, NOS2, NO, MHC-II, CD80, CD 86, and p-STAT1, but not in TRPC1-/- mice. Together, these results demonstrate an important role for TRPC1 in activation induced Ca2+ influx, and function in shaping macrophage polarization to M1-proinflammatory phenotype.

Efficient clearance of invading pathogen and the infiltrating cells by phagocytic cells are two key events that are required for successful elimination of a pathological insult. Clearance of the pathogen is usually achieved by phagocytosis and dead cells are cleared by a process called efferocytosis. While there is substantial information on various strategies employed by a number of pathogens to subvert phagocytosis, our knowledge about inhibition of efferocytosis by microbes is extremely limited. In that regard, we observed that, polymorphonuclear cells (PMNs) infected with Klebsiella pneumoniae (KPn), an opportunistic pathogen and a frequent cause of nosocomial infections leading to sepsis, were efferocytosed less than their uninfected counterparts. This inhibition correlated with the infection induced upregulation of “don’t eat me” molecules CD31 and CD47. KPn infection over time does decrease the amount of phosphatidylserine (PS), a well-studied "eat me" signal recognized by efferocytic cells, on the outer cell surface of infected PMNs. Increasing the amount of PS on the outer surface of KPn infected PMNs with treatment of N-ethylmaleimide (NEM), an inhibitor of the endogenous aminophospholipid flippase, prevented KPn induced inhibition of efferocytosis in infected PMNs. To our knowledge, this study is the first to show a modulatory effect of KPn infection on PMNs leading to inhibition of their efferocytic uptake by macrophages. This could have implications in acute and chronic airway inflammatory diseases such as pneumonia, sepsis, cystic fibrosis, and COPD.

Lyme disease, the most commonly reported vector borne illness in the United States, is caused by the spirochete Borrelia burgdorferi. Interestingly, B. burgdorferi has a propensity to adhere to host cells and tissues due to its ability to produce a large number of binding proteins. A common target for adherence is the glycoprotein fibronectin which is produced by host cells. Previous research has demonstrated that B. burgdorferi also has the ability to invade non-professional phagocytes in order to evade the immune response; however, the mechanism by which this occurs is still unidentified. In other bacteria, fibronectin-binding proteins are known to act as a bridge between host cell receptors and the bacteria to promote uptake by host cells; therefore, we hypothesized that one of these proteins plays a role in invasion by B. burgdorferi. One candidate for this role is BB0347, an outer-surface fibronectin-binding protein, because preliminary data suggest that it has multiple fibronectin binding domains similar to other characterized internalins. To analyze whether or not BB0347 aids in invasion, we have performed several experiments including internalization assays. Our preliminary data suggest that BB0347 may play a significant role in invading host cells via binding to fibronectin.

Combined antiretroviral therapeutic (ART) strategies have effectively increased the long-term survival of HIV-1 infected individuals. However, along with increased longevity comes age-related neurological disorders including a very high prevalence of HIV-1 associated neurocognitive disorders (HAND) including clinical manifestations and pathological features of Alzheimer’s disease (AD). However, the pathogenesis of HAND remains unclear, and little is known about how AD-like pathology is developed as a result of HIV-1 infection and/or long-term use of ART drugs. We postulate that neuronal endolysosome de-acidification leads to the co-pathogenesis of HAND and AD. Here, we used rat primary cultured neurons to test the effects of 14 separate ART drugs on endolysosome pH and amyloid beta (Aβ) protein generation. The 14 ART drugs tested included (1) the nucleoside reverse-transcriptase inhibitors zidovudine, abacavir, lamivudine, deoxythymidine and emtricitabine, (2) the nucleotide reverse-transcriptase inhibitors tenofovir and disoproxil, (3) the non-nucleoside reverse-transcriptase inhibitors efavirenz and nevirapine, (4) the protease inhibitors ritonavir, nelfinavir and darunavir, (5) the integrase inhibitors dolutegravir and elvitegravir, and (6) the booster (liver enzyme inhibitor) cobicistat. The ART drugs that de-acidified endolysosome pH also increased Aβ levels, whereas those ART drugs that acidified endolysosome pH decreased Aβ levels. Furthermore, an agent that acidifies endolysosomes (MLSA-1) blocked ART drug-induced increases in Aβ levels. Collectively, our data suggest endolysosome de-acidification plays an important role in ART drug-induced amyloidogenesis. (This work was supported by P30GM103329, and R01MH100972, and R01MH105329.)

Type III secretion (T3S) systems, common to many Gram- bacteria, act as a needle to deliver virulence factors directly into eukaryotic cells, often modulating host immunity. Multimers of YscF polymerize to form the surface-located T3S needle-like structure. We have previously shown that YscF has PAMP properties, acting as both a TLR2 and TLR4 agonist. We have also shown that modulation of this PAMP activity in vitro is located in the N terminus of YscF. We hypothesized that the N terminus of YscF contributes to Y. pestis survival within the eukaryotic host. We generated a 15 amino acid N terminal truncation of YscF (delta15YscF) expressed in Y. pestis. The truncation did not affect T3S system translocation; the delta15YscF Y. pestis strain was able to secrete Yops, albeit at a level lower than wild type (wt) Y. pestis. As expected, wt Y. pestis colonized the spleen and liver, whereas delta15YscF Y. pestis exhibited significantly impaired colonization in both organs. Mice infected with delta15YscF Y. pestis developed significantly more inflammation compared to wt Y. pestis. At 3 days p.i., delta15YscF spleen weights were significantly heavier and spleen inflammation was accompanied by a significant enhancement of macrophage and neutrophil migration compared to wt mice. These data support a role for the N. terminus of YscF contributing to host bacterial clearance. Truncating the N terminus may result in a PAMP which is thereby unmasked and able to induce an inflammatory response.

Helminth parasitic infections are associated with eosinophilia and development of myeloid cells of the alternatively activated macrophage (AAM) phenotype, but an absence of neutrophilia. However, specific AAM associated mechanisms, if any, which determine the absence of neutrophils in chronic helminth infections, is unknown. We examined the function of galectins, mammalian beta galactose-binding lectins, in a murine model for neurocysticercosis (NCC) induced by intracranial inoculation of the helminth parasite Mesocestoides corti. Parasite infection induced multiple galectins, among which galectin-3 was the first as to be upregulated as well as at a relatively high level in AAMs. Parasite-infected Galectin-3-/- mice displayed significantly increased susceptibility to parasite infection, despite a similar parasite burden in the central nervous system (CNS) compared to infected wild-type (WT) mice. The accumulation of macrophages, dendritic cells, γδ T cells, αβ T cells, or B cells in the brains of parasite-infected Galectin-3-/- mice was similar to the WT mice. Moreover, AAMs which play a profound protective role in murine NCC were observed at a similar level in infected WT and Galectin-3-/- mice. Instead, the parasite-infected Galectin-3-/- mice exhibited a massive neutrophilia in the CNS, and an increase in neutrophil-associated neuroinflammation. In a resolving inflammation, the dead/dying host cells are effectively cleared by phagocytic cells by a process termed as efferocytosis to prevent the potential tissue damage. In order to examine if the neutrophilia observed in Galectin-3-/- mice was due to a defect in efferocytosis, neutrophil clearance ability of Galectin-3- deficient and competent AAMs was compared. Interestingly, the Galectin-3-/- AAMs isolated from parasite infected mice, mice injected with IL-4, or macrophages differentiated in-vitro under the influence of IL-4, exhibited a defect in efferocytosis and clearance of neutrophils. These results suggest that Galectin-3 in AAMs play an important role in resolution of neutrophilia and inflammation, possibly by facilitating neutrophil turnover in helminth infection. This is the first report depicting the physiological function of Galectin-3 in helminth parasite infection of the CNS. The results can have implications not only in NCC but also in other neuroinflammatory diseases.

Neutrophil extracellular traps (NETs) constitute antimicrobial function of neutrophils but have also been linked to perpetuation of inflammation. Despite this evident physiological relevance mechanistic understanding of NET formation is poor. Here we show that Mincle a mammalian C-type lectin receptor regulates 28 NET formation in response to several activation stimuli in-vitro as well as in-vivo during pneumoseptic infection with Klebsiella pneumoniae. Importantly Mincle deficiency does not affect the ability of neutrophils to produce reactive oxygen species (ROS). Instead the attenuated NET formation in Mincle-/- 31 neutrophils correlates with an impaired autophagy activation in-vitro and in-vivo. We further demonstrate this Mincle/autophagy/NET axis is distinct from Syk/CARD9 and MAP kinase pathway. Altogether Mincle emerges as a central regulator of NET formation by controlling autophagy without Compromising ROS generation. Our study addresses a major challenge in the field by positing this pathway to be targeted for modulation of NETs while preserving ROS production an important innate immune defense.

Northeast North Dakota is highly agricultural with large amounts of acreage devoted to industrial scale farming. But lying within the expanse of crops are islands of forested areas. Since 2010, breeding populations of deer tick, Ixodes scapularis, have consistently been present in the larger (>200 hectares) 'forest islands'. Field infection rates of Borrelia burgdorferi (agent of Lyme disease) and Anaplasmosis phagocytophilum in North Dakota deer ticks have been relatively low (3 - 5%) compared to endemic regions to the east. Analyses of the mitochondrial DNA of deer ticks collected from different forest islands revealed a high haplotype diversity (Hd=0.80), suggesting that the forest islands were being colonized continuously from multiple sources. The likeliest host candidate to explain such pattern would be birds. Therefore, in 2014, birds were collected in northwest Minnesota - the geographically closest area of Lyme disease endemicity. Of 104 passerine birds examines, five (4.8%) tested positive by PCR and sequencing for Borrelia burgdorferi, supporting the hypothesis of avian introduction of infected ticks into North Dakota. Most of the small mammals captured in forest islands were either white-footed mice (Peromyscus - classic reservoir of Lyme disease) or red-backed voles (Myodes gapperi). Although both species were parasitized by immature deer ticks, larval l. scapularis infestation was significantly higher in mice than in voles. A small percentage (ca. 6%) of engorged ticks pulled from voles and mice tested positive by PCR for Borrelia and Anaplasma. To determine whether voles served as reservoirs for Lyme disease or as dilution hosts, a laboratory colony of red-backed voles was established. F-1 voles were injected with a North Dakota strain of B. burgdorferi s.s. and subsequently infested with larval deer ticks at 10, 20, and 40 days after injections. Engorged larvae molted to nymphs and were re-fed on naive laboratory mice. Voles remained highly infective to larval l. scapularis for 40 days and the resultant nymphs transmitted spirochetes to naive mice at rates comparable to nymphs fed on infected Peromyscus mice. Because of their abundance and ability to infect ticks for over a month, red-backed voles should be considered an important reservoir of Lyme disease in North Dakota.