Cellular Cytokine Responses Research Articles

Three doses of Sars-CoV-2 mRNA vaccine in older adults result in similar antibody responses but reduced cellular cytokine responses relative to younger adults

ObjectivesBooster COVID-19 vaccinations are used to protect the elderly, a group vulnerable to severe disease. We compared humoral and cellular immunity in older versus younger adults up to eight months after administering a BNT16b2 booster vaccine dose. Next, we analyzed the plasma levels of soluble T cell activation/exhaustion markers. MethodsHome-dwelling older adults (n = 68, median age 86) and younger healthcare workers (n = 35, median age 39), previously vaccinated with two doses of BNT162b2, were given a booster dose at ten months after the initial dose. Our analysis consisted of spike-specific IgG, neutralizing antibodies, memory B cells, IFN-γ and IL-2 secreting T cells and soluble T cell exhaustion/activation markers. ResultsFollowing the initial two doses, the elderly cohort exhibited lower humoral and IFN-γ responses compared to younger adults. The booster dose increased the humoral responses in both older and younger adults. At two months after the booster dose, older and younger vaccinees had comparable levels of antibodies and the responses were maintained up to 18 months. The younger cohort elicited an increase in the cellular response, while no increase was detected in the elderly. The elderly had higher plasma levels of soluble forms of the T cell activation/exhaustion markers CD25 and TIM-3, which inversely correlated with age and T-cell cytokine responses. This suggests that these markers may be related to the observed dysfunctional cellular cytokine response in older adults. However, both elderly and younger adults who experienced breakthrough infections after booster vaccination, elicited more robust humoral and IFN-γ responses. ConclusionsThe booster dose elicited neutralizing and spike-specific antibody responses in the elderly that were comparable with that of the younger cohort. However, the lack of a strong cellular cytokine response to the third dose in the elderly may explain their vulnerability to severe infection and may be a consequence of exhausted or senescent T cell responses. (https://clinicaltrials.gov/study/NCT04706390).

T-Helper 22 Cell Type Responses to Nickel in Contact Allergic Subjects Are Associated with T-Helper 1, T-Helper 2, and T-Helper 17 Cell Cytokine Profile Responses and Patch Test Reactivity

Introduction: Contact allergy to nickel (Ni) is a delayed-type hypersensitivity reaction mediated by Ni-reactive T cells producing the hallmark cytokines of several T-helper cell (Th) populations including IFN-γ (Th1), IL-4, IL-5 and IL-13 (Th2), and IL-17A (Th17). IL-22-expressing CD4+ cells, which could be either Th17 co-expressing IL-22 or Th22, expressing IL-22 in the absence of IL-17A, have also been found in Ni-provoked skin of allergic subjects. It has been unclear if Ni-reactive T cells consist of distinct Th cell type populations or if they secrete a mix of Th cell hallmark cytokines. The aim herein was to assess if cellular cytokine responses to Ni, in ex vivo-stimulated peripheral blood mononuclear cells (PBMCs) from Ni-allergic subjects, include not only Th1, Th2, and Th17 but also Th22 hallmark cytokines and to define if the cytokines are produced by distinct cell populations representing different Th profiles. Methods: PBMC from Ni-allergic subjects (n = 15) with different degrees of patch test reactivity and non-allergic controls (n = 5) were in vitro stimulated with Ni. Cytokine levels in PBMC supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) (IFN-γ, IL-2, IL-3, IL-5, IL-6, IL-13, IL-17A, IL-22, and IL-31). FluoroSpot was used to assess if individual Ni-reactive cells produced single, or combinations of, cytokines representing different Th profiles. Cytokine combinations analyzed were IL-17A/IL-22/IFN-γ, IL-5/IL-17A/IFN-γ, IL-13/IL-22/IFN-γ, and IL-5/IL-13. Results: IL-22 as well as all other cytokines measured by ELISA were induced by Ni at higher levels in PBMC from allergic versus non-allergic subjects, with higher levels being associated with stronger patch test reactivity. The levels of most Ni-induced cytokines were positively correlated with each other; IL-2 displayed the highest correlation with other cytokines and IL-6 the lowest. FluoroSpot analysis showed that Th signature cytokines, IFN-γ (Th1), IL-5 and IL-13 (Th2), IL-17A (Th17), and IL-22 (Th22), were almost exclusively produced by distinct cell populations. Conclusion: Distinct Th cell populations, including Ni-reactive cells displaying Th1, Th2, Th17, and Th22 cytokine profiles, are all increased in PBMC from Ni-allergic subjects and positively associated with patch test reactivity. The relevance of these different Th profile populations for the up- or down-regulation of inflammatory reactions in the skin of Ni-allergic subjects remains to be clarified.

Exploring in vitro expression and immune potency in mice using mRNA encoding the Plasmodium falciparum malaria antigen, CelTOS.

The secreted malarial protein, Cell-Traversal protein for Ookinetes and Sporozoites (CelTOS), is highly conserved among Plasmodium species, and plays a role in the invasion of mosquito midgut cells and hepatocytes in the vertebrate host. CelTOS was identified as a potential protective antigen based on a proteomic analysis, which showed that CelTOS stimulated significant effector T cells producing IFN-γ in peripheral blood mononuclear cells (PBMCs) from radiation attenuated sporozoite-immunized, malaria-naïve human subjects. In a rodent malaria model, recombinant full-length CelTOS protein/adjuvant combinations induced sterile protection, and in several studies, functional antibodies were produced that had hepatocyte invasion inhibition and transmission-blocking activities. Despite some encouraging results, vaccine approaches using CelTOS will require improvement before it can be considered as an effective vaccine candidate. Here, we report on the use of mRNA vaccine technology to induce humoral and cell-mediated immune responses using this antigen. Several pfceltos encoding mRNA transcripts were assessed for the impact on protein translation levels in vitro. Protein coding sequences included those to evaluate the effects of signal sequence, N-glycosylation on translation, and of nucleoside substitutions. Using in vitro transfection experiments as a pre-screen, we assessed the quality of the expressed CelTOS target relative to the homogeneity, cellular localization, and durability of expression levels. Optimized mRNA transcripts, which demonstrated highest protein expression levels in vitro were selected for encapsulation in lipid nanoparticles (LNP) and used to immunize mice to assess for both humoral and cellular cytokine responses. Our findings indicate that mRNA transcripts encoding pfceltos while potent for inducing antigen-specific cellular cytokine responses in mice, were less able to mount PfCelTOS-specific antibody responses using a two-dose regimen. An additional booster dose was needed to overcome low seroconversion rates in mice. With respect to antibody fine specificities, N-glycosylation site mutated immunogens yielded lower immune responses, particularly to the N-terminus of the molecule. While it remains unclear the impact on CelTOS antigen as immunogen, this study highlights the need to optimize antigen design for vaccine development.

Onchocerca volvulus-specific antibody and cellular responses in onchocerciasis patients treated annually with ivermectin for 30 years and exposed to parasite transmission in central Togo.

BackgroundAnnual mass drug administrations (MDA) of ivermectin will strongly reduce Onchocerca volvulus microfilariae (mf) in the skin and in the onchocerciasis patients’ eyes. Ivermectin treatment will also affect the expression of immunity in patients, such that activated immune defenses may help control and contribute to clearance of mf of O. volvulus. Longitudinal surveys are a prerequisite to determining the impact of ivermectin on the status of anti-parasite immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist.Methodology/Principal findingsOnchocerciasis patients were treated annually with ivermectin and their Onchocerca volvulus antigen (OvAg) specific IgG and cellular responses were investigated before and at 30 years post initial ivermectin treatment (30yPT).Repeated annual ivermectin treatments eliminated persisting O. volvulus microfilariae (mf) from the skin of patients and abrogated patent infections. The OvAg-specific IgG1 and IgG4 responses were diminished at 30yPT to the levels observed in endemic controls. Prior to starting ivermectin treatment, OvAg-induced cellular productions of IL-10, IFN-γ, CCL13, CCL17 and CCL18 were low in patients, and at 30yPT, cellular cytokine and chemokine responses increased to the levels observed in endemic controls. In contrast, mitogen(PHA)- induced IL-10, IFN-γ, CCL17 and CCL18 cellular production was diminished. This divergent response profile thus revealed increased parasite antigen-specific but reduced polyclonal cellular responsiveness in patients. The transmission of O. volvulus continued at the patients’ location in the Mô river basin in central Togo 2018 and 2019 when 0.58% and 0.45%, respectively, of Simulium damnosum s.l. vector blackflies carried O. volvulus infections.Conclusions/SignificanceRepeated annual ivermectin treatment of onchocerciasis patients durably inhibited their patent O. volvulus infections despite ongoing low-level parasite transmission in the study area. Repeated MDA with ivermectin affects the expression of immunity in patients. O. volvulus parasite-specific antibody levels diminished to levels seen in infection-free endemic controls. With low antibody levels, antibody-dependent cellular cytotoxic responses against tissue-dwelling O. volvulus larvae will weaken. O. volvulus antigen inducible cytokine and chemokine production increased in treated mf-negative patients, while their innate responsiveness to mitogen declined. Such lower innate responsiveness in elderly patients could contribute to reduced adaptive immune responses to parasite infections and vaccines. On the other hand, increased specific cellular chemokine responses in mf-negative onchocerciasis patients could reflect effector cell activation against tissue invasive larval stages of O. volvulus. The annual Simulium damnosum s.l. biting rate observed in the Mô river basin was similar to levels prior to initiation of MDA with ivermectin, and the positive rtPCR results reported here confirm ongoing O. volvulus transmission.

Streptococcus agalactiae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) elicits multiple cytokines from human cells and has a minor effect on bacterial persistence in the murine female reproductive tract

ABSTRACT Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH), encoded by gapC, is a glycolytic enzyme that is associated with virulence and immune-mediated protection. However, the role of GAPDH in cellular cytokine responses to S. agalactiae, bacterial phagocytosis and colonization of the female reproductive tract, a central host niche, is unknown. We expressed and studied purified recombinant GAPDH (rGAPDH) of S. agalactiae in cytokine elicitation assays with human monocyte-derived macrophage, epithelial cell, and polymorphonuclear leukocyte (PMN) co-culture infection models. We also generated a S. agalactiae mutant that over-expresses GAPDH (oeGAPDH) from gapC using a constitutively active promoter, and analyzed the mutant in murine macrophage antibiotic protection assays and in virulence assays in vivo, using a colonization model that is based on experimental infection of the reproductive tract in female mice. Human cell co-cultures produced interleukin (IL)-1β, IL-6, macrophage inflammatory protein (MIP)-1, tumor necrosis factor (TNF)-α and IL-10 within 24 h of exposure to rGAPDH. PMNs were required for several of these cytokine responses. However, over-expression of GAPDH in S. agalactiae did not significantly affect measures of phagocytic uptake compared to an empty vector control. In contrast, oeGAPDH-S. agalactiae showed a small but statistically significant attenuation for persistence in the reproductive tract of female mice during the chronic phase of infection (10–28 days post-inoculation), relative to the vector control. We conclude that S. agalactiae GAPDH elicits production of multiple cytokines from human cells, and over-expression of GAPDH renders the bacterium more susceptible to host clearance in the female reproductive tract. One-sentence summary: This study shows Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase, an enzyme that functions in glycolysis, gluconeogenesis and virulence, modifies phagocytosis outcomes, including cytokine synthesis, and affects bacterial persistence in the female reproductive tract.

Targeted Transcriptomics of Frog Virus 3 in Infected Frog Tissues Reveal Non-Coding Regulatory Elements and microRNAs in the Ranaviral Genome and Their Potential Interaction with Host Immune Response.

BackgroundFrog Virus 3 (FV3) is a large dsDNA virus belonging to Ranaviruses of family Iridoviridae. Ranaviruses infect cold-blood vertebrates including amphibians, fish and reptiles, and contribute to catastrophic amphibian declines. FV3 has a genome at ~105 kb that contains nearly 100 coding genes and 50 intergenic regions as annotated in its reference genome. Previous studies have mainly focused on coding genes and rarely addressed potential non-coding regulatory role of intergenic regions.ResultsUsing a whole transcriptomic analysis of total RNA samples containing both the viral and cellular transcripts from FV3-infected frog tissues, we detected virus-specific reads mapping in non-coding intergenic regions, in addition to reads from coding genes. Further analyses identified multiple cis-regulatory elements (CREs) in intergenic regions neighboring highly transcribed coding genes. These CREs include not only a virus TATA-Box present in FV3 core promoters as in eukaryotic genes, but also viral mimics of CREs interacting with several transcription factors including CEBPs, CREBs, IRFs, NF-κB, and STATs, which are critical for regulation of cellular immunity and cytokine responses. Our study suggests that intergenic regions immediately upstream of highly expressed FV3 genes have evolved to bind IRFs, NF-κB, and STATs more efficiently. Moreover, we found an enrichment of putative microRNA (miRNA) sequences in more than five intergenic regions of the FV3 genome. Our sequence analysis indicates that a fraction of these viral miRNAs is targeting the 3’-UTR regions of Xenopus genes involved in interferon (IFN)-dependent responses, including particularly those encoding IFN receptor subunits and IFN-regulatory factors (IRFs).ConclusionsUsing the FV3 model, this study provides a first genome-wide analysis of non-coding regulatory mechanisms adopted by ranaviruses to epigenetically regulate both viral and host gene expressions, which have co-evolved to interact especially with the host IFN response.

Elucidating Carbohydrate-Protein Interactions Using Nanoparticle-Based Approaches.

Carbohydrates are present on every living cell and coordinate important processes such as self/non-self discrimination. They are amongst the first molecular determinants to be encountered when cellular interactions are initiated. In particular, they resemble essential molecular fingerprints such as pathogen-, danger-, and self-associated molecular patterns guiding key decision-making in cellular immunology. Therefore, a deeper understanding of how cellular receptors of the immune system recognize incoming particles, based on their carbohydrate signature and how this information is translated into a biological response, will enable us to surgically manipulate them and holds promise for novel therapies. One approach to elucidate these early recognition events of carbohydrate interactions at cellular surfaces is the use of nanoparticles coated with defined carbohydrate structures. These particles are captured by carbohydrate receptors and initiate a cellular cytokine response. In the case of endocytic receptors, the capturing enables the engulfment of exogenous particles. Thereafter, the particles are sorted and degraded during their passage in the endolysosomal pathway. Overall, these processes are dependent on the nature of the endocytic carbohydrate receptors and consequently reflect upon the carbohydrate patterns on the exogenous particle surface. This interplay is still an under-studied subject. In this review, we summarize the application of nanoparticles as a promising tool to monitor complex carbohydrate-protein interactions in a cellular context and their application in areas of biomedicine.

Selective inflammatory propensities in adopted adolescents institutionalized as infants

This study examined whether early life adversity (ELA) limited to infancy was associated with an increase in circulating levels of proinflammatory cytokines and cellular cytokine responses to three stimulants [lipopolysaccharide (LPS), phytohemagglutinin (PHA), and phorbol myristate acetate plus ionomycin (PMA/IO)]. Participants were previously institutionalized (PI) youth (N = 45, 56 % female) who had spent their first years in institutional care (e.g., orphanages, baby homes) before being adopted into well-resourced homes (median age at adoption = 13 mos) and non-adopted comparisons (NA; N = 38, 55 % female). Their age range was 13.3–21.2 years (M = 16.3 years). This analysis followed up an earlier report on these youth (Reid et al., 2019a) that identified an increase in terminally differentiated CD8 + CD57 T cells among the PI relative to the NA youth. Cytokine levels in circulation were not highly correlated and thus examined separately. PI youth had higher circulating levels of Tumor Necrosis Factor-alpha (TNFα), but not Interleukin-1β (IL-1β) or Interleukin-6 (IL-6). Cytokine responses to in vitro activation within each stimulant condition were highly correlated and were thus combined to generate an index of the inflammatory reaction to each stimulant. Using Multivariate Analysis of Covariance, there was a highly significant multivariate effect of group, which was carried primarily by the PMA/IO condition, with PI youth exhibiting a larger inflammatory response than NA youth. Tests of mediation showed that both the early rearing effects on circulating TNFα and the composite inflammatory index of PMA/IO responsiveness were mediated in the statistical model by the percentage of CD8 + CD57+ TEMRA cells in circulation, a marker of replicative senescence in T cells. Sex differences were also found in circulating levels of IL-6 and TNFα, with males having higher levels than females.

Immune responses to SARS-CoV-2 in three children of parents with symptomatic COVID-19

Compared to adults, children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have predominantly mild or asymptomatic infections, but the underlying immunological differences remain unclear. Here, we describe clinical features, virology, longitudinal cellular, and cytokine immune profile, SARS-CoV-2-specific serology and salivary antibody responses in a family of two parents with PCR-confirmed symptomatic SARS-CoV-2 infection and their three children, who tested repeatedly SARS-CoV-2 PCR negative. Cellular immune profiles and cytokine responses of all children are similar to their parents at all timepoints. All family members have salivary anti-SARS-CoV-2 antibodies detected, predominantly IgA, that coincide with symptom resolution in 3 of 4 symptomatic members. Plasma from both parents and one child have IgG antibody against the S1 protein and virus-neutralizing activity detected. Using a systems serology approach, we demonstrate higher levels of SARS-CoV-2-specific antibody features of these family members compared to healthy controls. These data indicate that children can mount an immune response to SARS-CoV-2 without virological confirmation of infection, raising the possibility that immunity in children can prevent the establishment of SARS-CoV-2 infection. Relying on routine virological and serological testing may not identify exposed children, with implications for epidemiological and clinical studies across the life-span.

IMMU-34. LANDSCAPE OF GLIOBLASTOMA-ASSOCIATED IMMUNITY BY INTEGRATION OF scRNA-SEQ AND SPATIAL TRANSCRIPTOMICS

Abstract The diversity of molecular states and cellular plasticity of immune cells within the glioblastoma (GBM) environment remain poorly investigated. Here, we conduct deep transcriptional profiling of lymphoid and myeloid cell populations by scRNA-sequencing, map potential cellular interactions and cytokine responses that lead to the dysfunctional and exhausted phenotype of T cells. We identified Interleukin 10 (IL-10) response during T cell activation, which lead to a dysfunctional state of T cells. By the use of a novel method: The nearest functionally connected neighbor (NFCN), an in-silico model to explore cell-cell interaction, the dysfunctional/exhausted phenotype was found to be driven by subset of myeloid cells defined by high expression of HMOX1. By using spatial transcriptomic RNA-sequencing, we identified a correlation between T cell exhaustion and colocalized mesenchymal gene expression. We found that HMOX1 expressing myeloid cells occupying regions marked by T cell exhaustion. Using a human neocortical slice model with myeloid cell depletion we confirmed the functional interaction of myeloid and lymphoid cell leading to the dysfunctional state of T cells. A comprehensive understanding of cellular states and plasticity of lymphoid cells in GBM aids in providing successful immunotherapeutic approaches.

Increased inflammation and suboptimal infants outcomes in women with a history of Major Depressive Disorder from the Psychiatry Research and Motherhood-Depression (PRAM-D) Study

This study examined whether early life adversity (ELA) limited to infancy was associated with an increase in circulating levels of proinflammatory cytokines and cellular cytokine responses to three stimulants [lipopolysaccharide (LPS), phytohemagglutinin (PHA), and phorbol myristate acetate plus ionomycin (PMA/IO)]. Participants were previously institutionalized (PI) youth (N = 45, 56 % female) who had spent their first years in institutional care (e.g., orphanages, baby homes) before being adopted into well-resourced homes (median age at adoption = 13 mos) and non-adopted comparisons (NA; N = 38, 55 % female). Their age range was 13.3–21.2 years (M = 16.3 years). This analysis followed up an earlier report on these youth (Reid et al., 2019a) that identified an increase in terminally differentiated CD8 + CD57 T cells among the PI relative to the NA youth. Cytokine levels in circulation were not highly correlated and thus examined separately. PI youth had higher circulating levels of Tumor Necrosis Factor-alpha (TNFα), but not Interleukin-1β (IL-1β) or Interleukin-6 (IL-6). Cytokine responses to in vitro activation within each stimulant condition were highly correlated and were thus combined to generate an index of the inflammatory reaction to each stimulant. Using Multivariate Analysis of Covariance, there was a highly significant multivariate effect of group, which was carried primarily by the PMA/IO condition, with PI youth exhibiting a larger inflammatory response than NA youth. Tests of mediation showed that both the early rearing effects on circulating TNFα and the composite inflammatory index of PMA/IO responsiveness were mediated in the statistical model by the percentage of CD8 + CD57+ TEMRA cells in circulation, a marker of replicative senescence in T cells. Sex differences were also found in circulating levels of IL-6 and TNFα, with males having higher levels than females.

Ex vivo enrichment of PRAME antigen-specific T cells for adoptive immunotherapy using CD137 activation marker selection.

ObjectiveAdoptive immunotherapy with ex vivo expanded tumor‐specific T cells has potential as anticancer therapy. Preferentially expressed antigen in melanoma (PRAME) is an attractive target overexpressed in several cancers including melanoma and acute myeloid leukaemia (AML), with low expression in normal tissue outside the gonads. We developed a GMP‐compliant manufacturing method for PRAME‐specific T cells from healthy donors for adoptive immunotherapy.MethodsMononuclear cells were pulsed with PRAME 15‐mer overlapping peptide mix. After 16 h, activated cells expressing CD137 were isolated with immunomagnetic beads and cocultured with irradiated CD137neg fraction in medium supplemented with interleukin (IL)‐2, IL‐7 and IL‐15. Cultured T cells were restimulated with antigen‐pulsed autologous cells after 10 days. Cellular phenotype and cytokine response following antigen re‐exposure were assessed with flow cytometry, enzyme‐linked immunospot (ELISPOT) and supernatant cytokine detection. Detailed phenotypic and functional analysis with mass cytometry and T‐cell receptor (TCR) beta clonality studies were performed on selected cultures.ResultsPRAME‐stimulated cultures (n = 10) had mean expansion of 2500‐fold at day 18. Mean CD3+ percentage was 96% with CD4:CD8 ratio of 4:1. Re‐exposure to PRAME peptide mixture showed enrichment of CD4 cells expressing interferon (IFN)‐γ (mean: 12.2%) and TNF‐α (mean: 19.7%). Central and effector memory cells were 23% and 72%, respectively, with 24% T cells expressing PD1. Mass cytometry showed predominance of Th1 phenotype (CXCR3+/CCR4neg/CCR6neg/Tbet+, mean: 73%) and cytokine production including IL‐2, IL‐4, IL‐8, IL‐13 and GM‐CSF (2%, 6%, 8%, 4% and 11%, respectively).ConclusionPRAME‐specific T cells for adoptive immunotherapy were enriched from healthy donor mononuclear cells. The products were oligoclonal, exhibited Th1 phenotype and produced multiple cytokines.

Lineage and Spatial Mapping of Glioblastoma-associated Immunity

The diversity of molecular states and cellular plasticity of immune cells within the glioblastoma (GBM) environment remain poorly investigated. Here, we performed deep transcriptional profiling of lymphoid and myeloid cell populations by scRNA-sequencing, and mapped potential cellular interactions and cytokine responses that lead to the dysfunctional and exhausted phenotype of T cells. We identified the Interleukin 10 (IL-10) response during T cell activation as leading to a dysfunctional state in T cells. By the use of a novel method, the nearest functionally connected neighbor (NFCN), an in-silico model to explore cell-cell interactions, the dysfunctional/exhausted phenotype was found to be driven by a subset of myeloid cells defined by high expression of HMOX1. By using spatial transcriptomic RNA-sequencing, we identified a correlation between T cell exhaustion and colocalized mesenchymal gene expression also found that HMOX1 expressing myeloid cells occupied regions marked by T cell exhaustion. Using a human neocortical slice model with myeloid cell depletion we confirmed the functional interaction of myeloid and lymphoid cells, leading to the dysfunctional state of T cells. A comprehensive understanding of cellular states and plasticity of lymphoid cells in GBM aids in providing successful immunotherapeutic approaches.

Metagenomic Characterization of Influenza Virus: Bacteria Super-Infections Associated With Death and Survival

Abstract Bacterial super-infections (BSIs) increase morbidity and mortality during influenza epidemics and pandemics, but not all strains of influenza virus uniformly increase host susceptibility to BSIs. Infection of mice with a swine virus isolate, A/swine/Texas/4199-2/98-H3N2 (TX98), did not significantly predispose the host to BSI mortality regardless of whether the BSI was caused by S aureus, S pneumoniae, or S pyogenes. Comparison of this survival phenotype with the laboratory influenza virus strain A/Puerto Rico/3/1934-H1N1, which demonstrates lethal synergism, showed that cellular recruitment profiles and cytokine responses differ between the two viruses in a manner that implicates host responses against the invading virus as a contributor to secondary infection severity. To better understand host, viral, and bacterial responses underlying disparate outcomes, we utilized metagenomic analysis of 559 genes, the NanoString nCounter Immunology Panel-Plus kit and FFPE mouse lungs from a published BSI time course. Results show an overall increased level of gene expression during BSIs associated with survival when compared to peak viral titers. Early viral clearance and the presence of S pyogenes in the lungs of TX98-infected lungs 24 hours after BSI was confirmed. Host responses tied to differences in early viral detection and clearance consisted of RIG-I, OAS, TLR3, TLR8, and TLR9. Key changes were noted in the expression of type I and II interferons, complement proteins, cytokines, chemokines, Fc receptors, scavenger receptors, the immunoproteasome, and genes associated with T, B, Treg, and NK cell activation. Interestingly, there was no significant increase in host antimicrobial peptides during BSIs associated with survival while CAMP and Nos2 were significantly increased 24 hours prior to death in lethal BSIs. To our knowledge, this is the first side-by-side metagenomics study of influenza BSIs associated with death and survival. Results offer mechanistic insight into clinical outcomes.

Effects of Gold Nanoparticles with Different Surface Charges on Cellular Internalization and Cytokine Responses in Monocytes

Surface modification of gold nanoparticles (AuNPs) affects the interactions between the AuNPs and biological systems. To use AuNPs in biomedical applications, an investigation of how different surface charges of AuNPs affect the internalization mechanisms and functions of monocytes is needed. In this study, U937 cells were exposed to AuNPs with positive (AuNPs+ve) and negative (AuNPs-ve) surface charges. Cellular responses to the AuNPs, including cell uptake pathways, cell activation and cytokine production, were analysed. The results indicated different responses to AuNPs-ve and AuNPs+ve in outcomes including internalization pathways, cell activation and cytokine production levels. Interestingly, the results suggested that AuNPs+ve tended to enter the cells via macropinocytosis and strongly elicited a cellular response of IL-6 production via NF-kB activation, whereas the AuNPs-ve suppressed IL-6 production. Therefore, these results provide essential information for designing nanoparticles and may contribute to possible future nanomedicine applications, such as drug delivery and vaccination.

Understanding Leukocyte Recruitment in Murine Ozone‐Induced Lung Inflammation

Ozone, a highly reactive air pollutant, has been linked to a variety of acute and chronic respiratory diseases in humans. Health Canada guidelines suggest acute (1 h) ozone exposures should not exceed 120 ppb. Studies exposing mice to much higher levels of ozone (2 ppm) suggest significant recruitment of lung neutrophils and macrophages at 24 h. However, to our knowledge, no studies have been conducted at ambient ozone concentrations. Daytime “ground” ozone levels can easily reach 50 ppb. We report that ambient ozone, at 50 ppb, induces alveolar cell death, leukocyte recruitment and has the ability to cause lung damage and thus is of relevance to public health. We hypothesized that CX3CR1 macrophages are protective in murine ozone‐induced lung inflammation and mediate lung neutrophil recruitment. We exposed wild‐type and CX3CR1‐null mice to 50 ppb ozone or filtered air for 2 hours, and collected peripheral blood, lung vascular perfusate and bronchoalveolar lavage at 0 h, 6 h and 22 h after exposure. Our preliminary data suggest alveolar macrophage, epithelial and endothelial cell toxicity, alveolar and systemic nuclear chromatin and actin deposits, with vascular neutrophil recruitment being observed as early as 0 h, 6 h and 22 h after ozone exposure. Moreover, absence of CX3CR1 leads to an exaggerated inflammatory response marked by enhanced neutrophil recruitment. We aim to further characterize the cellular metabolic and cytokine responses in our model, which will allow us to better understand how and why leukocytes are recruited in ozone‐induced lung inflammation, and will guide development of protective health strategies and environmental standards.Support or Funding InformationFedoruk Centre and Innovation SaskatchewanThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inflammatory and regulatory CCL and CXCL chemokine and cytokine cellular responses in patients with patent Mansonella perstans filariasis.

Mansonella perstans (Mp) filariasis is present in large populations in sub-Saharan Africa, and to what extent patent Mp infection modulates the expression of immunity in patients, notably their cellular cytokine and chemokine response profile, remains not well known. We studied the spontaneous and inducible cellular production of chemokines (C-X-C motif) ligand 9 (CXCL9) [monokine induced by interferon (IFN)-γ (MIG)], CXCL-10 [inducible protein (IP)-10], chemokine (C-C motif) ligand 24 (CCL24) (eotaxin-2), CCL22 [macrophage-derived chemokine (MDC)], CCL13 [monocyte chemotactic protein-4 (MCP-4)], CCL18 [pulmonary and activation-regulated chemokine (PARC)], CCL17 [thymus- and activation-regulated chemokine (TARC)] and interleukin (IL)-27 in mansonelliasis patients (Mp-PAT) and mansonelliasis-free controls (CTRL). Freshly isolated peripheral mononuclear blood cells (PBMC) were stimulated with helminth, protozoan and bacterial antigens and mitogen [phytohaemagglutinin (PHA)]. PBMC from Mp-PAT produced spontaneously (without antigen stimulation) significantly higher levels of eotaxin-2, IL-27, IL-8, MCP-4 and MDC than cells from CTRL, while IFN-γ-IP-10 was lower in Mp-PAT. Helminth antigens activated IL-27 and MCP-4 only in CTRL, while Ascaris antigen, Onchocerca antigen, Schistosoma antigen, Entamoeba antigen, Streptococcus antigen, Mycobacteria antigen and PHA stimulated MIG release in CTRL and Mp-PAT. Notably, Entamoeba antigen and PHA strongly depressed (P<0·0001) eotaxin-2 (CCL24) production in both study groups. Multiple regression analyses disclosed in Mp-PAT and CTRL dissimilar cellular chemokine and cytokine production levels being higher in Mp-PAT for CCL24, IL-27, IL-8, MCP-4, MDC and PARC (for all P<0·0001), at baseline (P<0·0001), in response to Entamoeba histolytica strain HM1 antigen (EhAg) (P<0·0001), Onchocerca volvulus adult worm-derived antigen (OvAg) (P=0·005), PHA (P<0·0001) and purified protein derivative (PPD) (P<0·0001) stimulation. In Mp-PAT with hookworm co-infection, the cellular chemokine production of CXCL10 (IP-10) was diminished. In summary, the chemokine and cytokine responses in Mp-PAT were in general not depressed, PBMC from Mp-PAT produced spontaneously and selectively inducible inflammatory and regulatory chemokines and cytokines at higher levels than CTRL and such diverse and distinctive reactivity supports that patent M. perstans infection will not polarize innate and adaptive cellular immune responsiveness in patients.

Interplay between Cellular Metabolism and Cytokine Responses during Viral Infection.

Metabolism and immune responses are two fundamental biological processes that serve to protect hosts from viral infection. As obligate intracellular pathogens, viruses have evolved diverse strategies to activate metabolism, while inactivating immune responses to achieve maximal reproduction or persistence within their hosts. The two-way virus-host interaction with metabolism and immune responses choreograph cytokine production via reprogramming metabolism of infected cells/hosts. In return, cytokines can affect the metabolism of virus-infected and bystander cells to impede viral replication processes. This review aims to summarize our current understanding of the cross-talk between metabolic reprogramming and cytokine responses, and to highlight future potential research topics. Although the focus is placed on viral pathogens, relevant findings from other microbes are integrated to provide an overall picture, particularly when corresponding information on viral infection is lacking.

A Synthetic Micro-Consensus DNA Vaccine Generates Comprehensive Influenza A H3N2 Immunity and Protects Mice Against Lethal Challenge by Multiple H3N2 Viruses.

Influenza A H3N2 viruses circulate globally, leading to substantial morbidity and mortality. Commercially available, antigen-matched influenza vaccines must be updated frequently to match dynamic sequence variability in immune epitopes, especially within viral influenza A H3N2 hemagglutinin (H3). In an effort to create comprehensive immune responses against H3N2, four micro-consensus antigens were designed to mimic the sequence and antigenic diversity of H3. Synthetic plasmid DNA constructs were developed to express each micro-consensus immunogen and combined into a multi-antigen DNA vaccine cocktail, pH3HA. Facilitated delivery of pH3HA via intramuscular electroporation in mice induced comprehensive, potent humoral responses against diverse seasonal H3N2 viruses that circulated between 1968 and the present. Vaccination with pH3HA also induced an antigen-specific cellular cytokine response. Mice immunized with pH3HA were protected against lethal challenge using two distinct H3N2 viruses, highlighting the heterologous protection afforded by synthetic micro-consensus immunogens. These findings warrant further study of the DNA vaccine micro-consensus platform for broad protection against influenza viruses.

Immune Profiling of T Cells Infiltrating Vitreous Humor in Tubercular Uveitis

ABSTRACTPurpose: To assess cellular composition and local cytokine response in vitreous humor of tubercular uveitis.Methods: Cells were collected from vitreous cassettes and peripheral blood of 8 tubercular uveitis and 5 control subjects, undergoing vitrectomy and analyzed by flow cytometry for cellular composition, activation status, proinflammatory cytokine expression, and uptake of TLR9 ligand, CpG ODN 2216.Results: CD3 + T cells with equal proportion of CD4+ and CD8 + T cells formed major fraction of infiltrating cells. The vitreous humor showed higher expression of recent activation marker, CD69, and proinflammatory cytokines, IFN-γ and IL-17A, in CD4 + T cells as compared to peripheral blood. Lastly, intraocular CD4 + T cells showed reduced uptake of ODN 2216 than peripheral blood.Conclusions: Our results indicate that local antigenic stimuli trigger T cell infiltration and activation of CD4 + T cells that are hyporesponsive to TLR9 stimulation. These infiltrating T cells might be responsible in further aggravating ocular inflammation.