Immunoglobulin A antibody composition is sculpted to bind the self gut microbiome.

Despite being the most abundantly secreted immunoglobulin isotype, the pattern of reactivity of immunoglobulin A (IgA) antibodies toward each individual's own gut commensal bacteria still remains elusive. By colonizing germ-free mice with defined commensal bacteria, we found that the binding specificity of bulk fecal and serum IgA toward resident gut bacteria resolves well at the species level and has modest strain-level specificity. IgA hybridomas generated from lamina propria B cells of gnotobiotic mice showed that most IgA clones recognized a single bacterial species, whereas a small portion displayed cross-reactivity. Orally administered hybridoma-produced IgAs still retained bacterial antigen binding capability, implying the potential for a new class of therapeutic antibodies. Species-specific IgAs had a range of strain specificities. Given the distinctive bacterial species and strain composition found in each individual's gut, our findings suggest the IgA antibody repertoire is shaped uniquely to bind "self" gut bacteria.

Development of broadly neutralizing antibodies targeting the cytomegalovirus subdominant antigen gH.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that increases morbidity and mortality in immunocompromised individuals including transplant recipients and newborns. New anti-HCMV therapies are an urgent medical need for diverse patient populations. HCMV infection of a broad range of host tissues is dependent on the gH/gL/gO trimer and gH/gL/UL28/UL130/UL131A pentamer complexes on the viral envelope. We sought to develop safe and effective therapeutics against HCMV by generating broadly-neutralizing, human monoclonal antibodies (mAbs) from VelocImmune® mice immunized with gH/gL cDNA. Following high-throughput binding and neutralization screening assays, 11 neutralizing antibodies were identified with unique CDR3 regions and a high-affinity (KD 1.4-65 nM) to the pentamer complex. The antibodies bound to distinct regions within Domains 1 and 2 of gH and effectively neutralized diverse clinical strains in physiologically relevant cell types including epithelial cells, trophoblasts, and monocytes. Importantly, combined adminstration of mAbs with ganciclovir, an FDA approved antiviral, greatly limited virus dissemination. Our work identifies several anti-gH/gL mAbs and sheds light on gH neutralizing epitopes that can guide future vaccine strategies.

TMEM176B Regulates AKT/mTOR Signaling and Tumor Growth in Triple-Negative Breast Cancer.

TMEM176B is a member of the membrane spanning 4-domains (MS4) family of transmembrane proteins, and a putative ion channel that is expressed in immune cells and certain cancers. We aimed to understand the role of TMEM176B in cancer cell signaling, gene expression, cell proliferation, and migration in vitro, as well as tumor growth in vivo. We generated breast cancer cell lines with overexpressed and silenced TMEM176B, and a therapeutic antibody targeting TMEM176B. Proliferation and migration assays were performed in vitro, and tumor growth was evaluated in vivo. We performed gene expression and Western blot analyses to identify the most differentially regulated genes and signaling pathways in cells with TMEM176B overexpression and silencing. Silencing TMEM176B or inhibiting it with a therapeutic antibody impaired cell proliferation, while overexpression increased proliferation in vitro. Syngeneic and xenograft tumor studies revealed the attenuated growth of tumors with TMEM176B gene silencing compared with controls. We found that the AKT/mTOR signaling pathway was activated or repressed in cells overexpressing or silenced for TMEM176B, respectively. Overall, our results suggest that TMEM176B expression in breast cancer cells regulates key signaling pathways and genes that contribute to cancer cell growth and progression, and is a potential target for therapeutic antibodies.

Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera.

The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week.IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.

Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera.

The global COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVΔG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

Interleukin 22 disrupts pancreatic function in newborn mice expressing IL-23.

Neonatal inflammatory diseases are associated with severe morbidity, but the inflammatory factors underlying them and their potential effector mechanisms are poorly defined. Here we show that necrotizing enterocolitis in neonate mice is accompanied by elevation of IL-23 and IL-22 and decreased production of pancreatic enzymes. These phenotypes are mirrored in neonate mice overexpressing IL-23 in CX3CR1+ myeloid cells or in keratinocytes. The mice fail to grow and die prematurely, displaying systemic inflammation, nutrient malabsorption and decreased expression of intestinal and pancreatic genes mediating digestion and absorption of carbohydrates, proteins, and lipids. Germ-free environment improves, and genetic ablation of IL-22 restores normal growth in mice overexpressing IL-23. Mechanistically, IL-22 acts directly at the level of pancreatic acinar cells to decrease expression of the pancreas associated transcription factor 1a (PTF1a). These results show that augmented production of IL-23 and IL-22 in early life has a negative impact on pancreatic enzyme secretion and food absorption.

CD46 facilitates entry and dissemination of human cytomegalovirus.

Human cytomegalovirus (CMV) causes a wide array of disease to diverse populations of immune-compromised individuals. Thus, a more comprehensive understanding of how CMV enters numerous host cell types is necessary to further delineate the complex nature of CMV pathogenesis and to develop targeted therapeutics. To that end, we establish a vaccination strategy utilizing membrane vesicles derived from epithelial cells to generate a library of monoclonal antibodies (mAbs) targeting cell surface proteins in their native conformation. A high-throughput inhibition assay is employed to screen these antibodies for their ability to limit infection, and mAbs targeting CD46 are identified. In addition, a significant reduction of viral proliferation in CD46-KO epithelial cells confirms a role for CD46 function in viral dissemination. Further, we demonstrate a CD46-dependent entry pathway of virus infection in trophoblasts, but not in fibroblasts, highlighting the complexity of CMV entry and identifying CD46 as an entry factor in congenital infection.

Functional screening for anti-CMV biologics identifies a broadly neutralizing epitope of an essential envelope protein.

The prototypic ß-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies' epitope as an 'antigenic hot spot' critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.

CYLD Proteolysis Protects Macrophages from TNF-Mediated Auto-necroptosis Induced by LPS and Licensed by Type I IFN.

Tumor necrosis factor (TNF) induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages, including TLR4, TNF, and type I IFN receptors, are concurrently activated, but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase cylindromatosis (CYLD) in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While lipopolysaccharides (LPS)-induced necroptosis was abrogated in Tnf(-/-) macrophages, a soluble TNF antagonist was not able to do so in Tnf(+/+) macrophages, indicating that necroptosis occurs in a cell-autonomous manner. Surprisingly, TNF-mediated auto-necroptosis of macrophages requires type I IFN, which primes the expression of key necroptosis-signaling molecules, including TNFR2 and MLKL. Thus, the TNF necroptosis pathway is regulated by both negative and positive crosstalk.

Perfluoroalkyl substance serum concentrations and immune response to FluMist vaccination among healthy adults.

Perfluoroalkyl substances (PFAS) were shown to be immunotoxic in laboratory animals. There is some epidemiological evidence that PFAS exposure is inversely associated with vaccine-induced antibody concentration. We examined immune response to vaccination with FluMist intranasal live attenuated influenza vaccine in relation to four PFAS (perfluorooctanoate, perfluorononanoate, perfluorooctane sulfonate, perfluorohexane sulfonate) serum concentrations among 78 healthy adults vaccinated during the 2010-2011 influenza season. We measured anti-A H1N1 antibody response and cytokine and chemokine concentrations in serum pre-vaccination, 3 days post-vaccination, and 30 days post-vaccination. We measured cytokine, chemokine, and mucosal IgA concentration in nasal secretions 3 days post-vaccination and 30 days post-vaccination. Adults with higher PFAS concentrations were more likely to seroconvert after FluMist vaccination as compared to adults with lower PFAS concentrations. The associations, however, were imprecise and few participants seroconverted as measured either by hemagglutination inhibition (9%) or immunohistochemical staining (25%). We observed no readily discernable or consistent pattern between PFAS concentration and baseline cytokine, chemokine, or mucosal IgA concentration, or between PFAS concentration and change in these immune markers between baseline and FluMist-response states. The results of this study do not support a reduced immune response to FluMist vaccination among healthy adults in relation to serum PFAS concentration. Given the study's many limitations, however, it does not rule out impaired vaccine response to other vaccines or vaccine components in either children or adults.

Broadly neutralizing anti-influenza virus antibodies: enhancement of neutralizing potency in polyclonal mixtures and IgA backbones.

UNLABELLED: Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of "universal" influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during "universal" influenza virus vaccine design. IMPORTANCE: Influenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the emergence of pandemic strains, a new class of broadly neutralizing antibodies has been recently discovered and may be the key to developing a "universal" influenza virus vaccine. While much has been learned about the biology of these antibodies, most studies have focused only on monoclonal antibodies of IgG subtypes. However, the study of monoclonal antibodies often fails to capture the complexity of antibody functions that occur during natural polyclonal responses. Here, we provide the first detailed analyses of the biological activity of these antibodies in polyclonal contexts, comparing both IgG and IgA isotypes isolated from human donors. The striking differences observed in the functional properties of broadly neutralizing antibodies in polyclonal contexts will be essential for guiding design of "universal" influenza virus vaccines and therapeutics.

Human cytomegalovirus modulates monocyte-mediated innate immune responses during short-term experimental latency in vitro.

UNLABELLED: The ability of human cytomegalovirus (HCMV) to establish lifelong persistence and reactivate from latency is critical to its success as a pathogen. Here we describe a short-term in vitro model representing the events surrounding HCMV latency and reactivation in circulating peripheral blood monocytes that was developed in order to study the immunological consequence of latent virus carriage. Infection of human CD14(+) monocytes by HCMV resulted in the immediate establishment of latency, as evidenced by the absence of particular lytic gene expression, the transcription of latency-associated mRNAs, and the maintenance of viral genomes. Latent HCMV induced cellular differentiation to a macrophage lineage, causing production of selective proinflammatory cytokines and myeloid-cell chemoattractants that most likely play a role in virus dissemination in the host. Analysis of global cellular gene expression revealed activation of innate immune responses and the modulation of protein and lipid synthesis to accommodate latent HCMV infection. Remarkably, monocytes harboring latent virus exhibited selective responses to secondary stimuli known to induce an antiviral state. Furthermore, when challenged with type I and II interferon, latently infected cells demonstrated a blockade of signaling at the level of STAT1 phosphorylation. The data demonstrate that HCMV reprograms specific cellular pathways in monocytes, most notably innate immune responses, which may play a role in the establishment of, maintenance of, and reactivation from latency. The modulation of innate immune responses is likely a viral evasion strategy contributing to viral dissemination and pathogenesis in the host. IMPORTANCE: HCMV has the ability to establish a lifelong infection within the host, a phenomenon termed latency. We have established a short-term model system in human peripheral blood monocytes to study the immunological relevance of latent virus carriage. Infection of CD14(+) monocytes by HCMV results in the generation of latency-specific transcripts, maintenance of viral genomes, and the capacity to reenter the lytic cycle. During short-term latency in monocytes the virus initiates a program of differentiation to inflammatory macrophages that coincides with the modulation of cytokine secretion and specific cellular processes. HCMV-infected monocytes are hindered in their capacity to exert normal immunoprotective mechanisms. Additionally, latent virus disrupts type I and II interferon signaling at the level of STAT1 phosphorylation. This in vitro model system can significantly contribute to our understanding of the molecular and inflammatory factors that initiate HCMV reactivation in the host and allow the development of strategies to eradicate virus persistence.

Polymorphisms in inflammatory genes are associated with term small for gestational age and preeclampsia.

PROBLEM: Inflammatory biomarkers are associated with preeclampsia (PE) and poor fetal growth; however, genetic epidemiologic studies have been limited by reduced gene coverage and the exclusion of African American mothers. METHOD OF STUDY: Cases and controls (N = 1646) from a pregnancy cohort were genotyped for 503 tagSNPs in 40 genes related to inflammation. Gene-set analyses were stratified by race and were followed by a single SNP analysis within significant gene sets. RESULTS: Gene-level associations were found for IL6 and KLRD1 for term small for gestational age (SGA) among African Americans. LTA/TNF and TBX21 were associated with PE among European Americans. The strongest association was for PE among European Americans for an upstream regulator of TNF with RR = 1.8 (95% CI 1.1-2.7). CONCLUSION: Although previous studies have suggested null associations, increased tagging and stratification by genetic ancestry suggests important associations between IL6 and term SGA for African Americans, and a TNF regulator and PE among European Americans (N = 149).

Combinatorial cytokine code generates anti-viral state in dendritic cells.

The physiological function of the immune system and the response to therapeutic immunomodulators may be sensitive to combinatorial cytokine micro-environments that shape the responses of specific immune cells. Previous work shows that paracrine cytokines released by virus-infected human dendritic cells (DC) can dictate the maturation state of naïve DCs. To understand the effects of paracrine signaling, we systematically studied the effects of combinations cytokines in this complex mixture in generating an anti-viral state. After naïve DCs were exposed to either IFNß or to paracrine signaling released by DCs infected by Newcastle disease virus (NDV), microarray analysis revealed a large number of genes that were differently regulated by the DC-secreted paracrine signaling. In order to identify the cytokine mechanisms involved, we identified 20 cytokines secreted by NDV infected DCs for which the corresponding receptor gene is expressed in naïve DCs. By exposing cells to all combinations of 19 cytokines (leave-one-out studies), we identified five cytokines (IFNß, TNFα, IL-1ß, TNFSF15, and IL28) as candidates for regulating DC maturation markers. Subsequent experiments identified IFNß, TNFα, and IL1ß as the major contributors to this anti-viral state. This finding was supported by infection studies in vitro, by T-cell activation studies and by in vivo infection studies in mouse. Combination of cytokines can cause response states in DCs that differ from those achieved by the individual cytokines alone. These results suggest that the cytokine microenvironment may act via a combinatorial code to direct the response state of specific immune cells. Further elucidation of this code may provide insight into responses to infection and neoplasia as well as guide the development of combinatorial cytokine immunomodulation for infectious, autoimmune, and immunosurveillance-related diseases.

Plasma level of interferon γ induced protein 10 is a marker of sarcoidosis disease activity.

RATIONALE: Sarcoidosis is an idiopathic granulomatous disorder with heterogeneous clinical manifestations and variable prognosis. Monitoring disease activity is important to identify patients requiring treatment. Several cytokines have previously been shown to be elevated in the serum of patients with sarcoidosis and may be useful biomarkers of disease activity. OBJECTIVES: To identify novel biomarkers of sarcoidosis disease activity. To identify the relationship between plasma cytokines, disease severity and prognosis. METHODS: The study was approved by the institutional review board. Plasma concentration of 19 cytokines was measured in 112 subjects with chronic sarcoidosis and 52 matched controls, using the bead-based Milliplex xMAP multiplex technology. Plasma levels of individual cytokines were compared between the two groups, and between the groups with clinically active vs. inactive disease. Sensitivity, specificity and receiver operating characteristics curves were used to evaluate biomarker performance. Linear regression analyses were performed to identify associations between cytokine levels, pulmonary function tests and changes in pulmonary function. MEASUREMENTS AND MAIN RESULTS: Subjects with sarcoidosis had higher plasma levels of interferon gamma induced protein 10 (IP-10) and tumor necrosis factor α (TNFα). IP-10 had the highest sensitivity and specificity in identifying active disease. Higher levels of IP-10 and TNFα were associated with greater disease severity and better prognosis. CONCLUSIONS: IP-10 is a potentially useful biomarker of sarcoidosis and its severity.

Localized mucosal response to intranasal live attenuated influenza vaccine in adults.

BACKGROUND: Influenza virus infection is a major public health burden worldwide. Available vaccines include the inactivated intramuscular trivalent vaccine and, more recently, an intranasal live attenuated influenza vaccine (LAIV). The measure of successful vaccination with the inactivated vaccine is a systemic rise in immunoglobulin G (IgG) level, but for the LAIV no such correlate has been established. METHODS: Seventy-nine subjects were given the LAIV FluMist. Blood was collected prior to vaccination and 3 days and 30 days after vaccination. Nasal wash was collected 3 days and 30 days after vaccination. Responses were measured systemically and in mucosal secretions for cytokines, cell activation profiles, and antibody responses. RESULTS: Only 9% of subjects who received LAIV seroconverted, while 33% of patients developed at least a 2-fold increase in influenza virus-specific immunoglobulin A (IgA) antibodies in nasal wash. LAIV induced a localized inflammation, as suggested by increased expression of interferon-response genes in mucosal RNA and increased granulocyte colony-stimulating factor (G-CSF) and IP-10 in nasal wash. Interestingly, patients who seroconverted had significantly lower serum levels of G-CSF before vaccination. CONCLUSIONS: Protection by LAIV is likely provided through mucosal IgA and not by increases in systemic IgG. LAIV induces local inflammation. Seroconversion is achieved in a small fraction of subjects with a lower serum G-CSF level.

Mouse dendritic cell (DC) influenza virus infectivity is much lower than that for human DCs and is hemagglutinin subtype dependent.

We show that influenza A H1N1 virus infection leads to very low infectivity in mouse dendritic cells (DCs) in vitro compared with that in human DCs. This holds when H3 or H5 replaces H1 in recombinant viruses. Viruslike particles confirm the difference between mouse and human, suggesting that reduced virus entry contributes to lower mouse DC infectivity. Low infectivity of mouse DCs should be considered when they are used to study responses of DCs that are actually infected.

Type I interferon induction during influenza virus infection increases susceptibility to secondary Streptococcus pneumoniae infection by negative regulation of γδ T cells.

The majority of deaths following influenza virus infection result from secondary bacterial superinfection, most commonly caused by Streptococcus pneumoniae. Several models have been proposed to explain how primary respiratory viral infections exacerbate secondary bacterial disease, but the mechanistic explanations have been contradictory. In this study, mice were infected with S. pneumoniae at different days after primary influenza A (X31) virus infection. Our findings show that the induction of type I interferons (IFNs) during a primary nonlethal influenza virus infection is sufficient to promote a deadly S. pneumoniae secondary infection. Moreover, mice deficient in type I interferon receptor (IFNAR knockout [KO] mice) effectively cleared the secondary bacterial infection from their lungs, increased the recruitment of neutrophils, and demonstrated an enhanced innate expression of interleukin-17 (IL-17) relative to wild-type (WT) mice. Lung γδ T cells were responsible for almost all IL-17 production, and their function is compromised during secondary S. pneumoniae infection of WT but not IFNAR KO mice. Adoptive transfer of γδ T cells from IFNAR KO mice reduced the susceptibility to secondary S. pneumoniae infection in the lung of WT mice. Altogether, our study highlights the importance of type I interferon as a key master regulator that is exploited by opportunistic pathogens such as S. pneumoniae. Our findings may be utilized to design effective preventive and therapeutic strategies that may be beneficial for coinfected patients during influenza epidemics.

Estrogen mediates innate and adaptive immune alterations to influenza infection in pregnant mice.

Pregnancy is a leading risk factor for severe complications during an influenza virus infection. Women infected during their second and third trimesters are at increased risk for severe cardiopulmonary complications, premature delivery, and death. Here, we establish a murine model of aerosolized influenza infection during pregnancy. We find significantly altered innate antiviral responses in pregnant mice, including decreased levels of IFN-ß, IL-1α, and IFN-γ at early time points of infection. We also find reduced cytotoxic T cell activity and delayed viral clearance. We further demonstrate that pregnancy levels of the estrogen 17-ß-estradiol are able to induce key anti-inflammatory phenotypes in immune responses to the virus independently of other hormones or pregnancy-related stressors. We conclude that elevated estrogen levels result in an attenuated anti-viral immune response, and that pregnancy-associated morbidities occur in the context of this anti-inflammatory phenotype.

Granulocyte colony-stimulating factor protects mice during respiratory virus infections.

A burst in the production of pro-inflammatory molecules characterizes the beginning of the host response to infection. Cytokines, chemokines, and growth factors work in concert to control pathogen replication and activate innate and adaptive immune responses. Granulocyte colony-stimulating factor (G-CSF) mobilizes and activates hematopoietic cells from the bone marrow, and it has been shown to mediate the generation of effective immunity against bacterial and fungal infections. G-CSF is produced at high levels in the lungs during infection with influenza and parainfluenza viruses, but its role during these infections is unknown. Here we show that during infection of mice with a non-lethal dose of influenza or Sendai virus, G-CSF promotes the accumulation of activated Ly6G+ granulocytes that control the extent of the lung pro-inflammatory response. Remarkably, these G-CSF-mediated effects facilitate viral clearance and sustain mouse survival.