Antimicrobial Immunity Research Articles

Early molecular events in PAMP-triggered immunity

In plant innate immunity, the first line of microbial recognition leading to active defence responses relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs). This recognition leads to PAMP-triggered immunity (PTI). Despite the numerous PAMPs recognised by plants, only a handful of PRRs are characterised. For most, they correspond to transmembrane proteins with a ligand-binding ectodomain. PRRs interact with additional transmembrane proteins that act as signalling adapters or amplifiers to achieve full functionality. The crucial role of PRRs in anti-microbial immunity is demonstrated by the direct targeting of PRRs and their associated proteins by pathogenic virulence effectors.

Acanthamoebaspp. in Urine of Critically Ill Patients

<i>Acanthamoeba</i>spp. in Urine of Critically Ill Patients

Human defensins activate monocyte-derived dendritic cells, promote the production of proinflammatory cytokines, and up-regulate the surface expression of CD91

Defensins are endogenous defense peptides with well defined antimicrobial activity against a broad spectrum of pathogens including bacteria, fungi, viruses, and parasites.Several lines of evidence suggest that defensins might also contribute to the regulation of host innate and adaptive immunity, but their immunomodulatory functions are still poorly understood. Herein, we studied the impact of human defensins on multiple functions of DCs, which are a central player in all immune responses, bridging innate and adaptive immunity. We challenged DCs differentiated in vitro from human moDCs with HNP-1 alpha-defensin or HBD-1. HNP-1 and HBD-1 were chemotactic for moDCs. Both defensins promoted the activation and maturation of moDCs, as assessed by up-regulation of surface expression of the costimulatory molecules CD80, CD86, and CD40, the maturation marker CD83, and HLA-DR. HNP-1 and HBD-1 also enhanced the production of the proinflammatory cytokines TNF-alpha, IL-6, and IL-12p70 but did not affect the production of the regulatory cytokine IL-10. According to these stimulatory effects, HNP-1 and HBD-1 increased the allostimulatory activity of moDCs significantly. Finally, HNP-1 and HBD-1 promoted the up-regulation of CD91 on the DC surface. CD91 is a scavenger receptor involved in the recognition of multiple ligands including defensins, thus suggesting that defensins may amplify their own effects through the activation of an autocrine loop. Taken together, our observations may provide new insight into the immunomodulatory properties of human defensins and may aid the exploration of new therapeutic strategies to potentiate antimicrobial and antitumor immunity.

Mechanisms of costimulation

The immune system has the remarkable ability to defend against a diversity of microbial pathogens, yet not respond to self. The discovery of the T-cell antigen receptor (TCR) provided an explanation for the specificity and diversity of T-cell responses. Yet, functional studies of Lafferty and colleagues (1) suggested that antigen alone was not sufficient to drive the activation of naive T cells and led to the two signal concept of T-cell activation. According to this model, productive T-cell activation requires a first signal provided by the interaction of antigenic peptide/major histocompatibility complex (MHC) with the TCR and a second, antigen-independent co-signal, the ‘costimulatory signal’. In addition, seminal studies of Jenkins and Schwartz (2, 3) demonstrated that TCR-mediated activation of T cells in the absence of costimulation resulted in antigen-specific unresponsiveness (termed T-cell anergy), rendering the T cells unable to respond to subsequent exposure to antigen. Thus, costimulation was postulated to have a pivotal role in determining whether the outcome of T-cell encounter with antigen would be activation or anergy. The critical role of costimulation in regulating the immune response has given impetus to the study of costimulation and resulted in incredible growth of this field, since an understanding of costimulation is of fundamental and therapeutic interest. Early studies defined the function of the CD28 receptor as a stimulatory receptor for the activation of naive T cells (2), and identified the B7 family members, B7-1 (CD80) and B7-2 (CD86) as its ligands (5–7). The interaction between CD28 and B7-1/B7-2 fulfilled many of the requirements for the costimulatory signal postulated by Lafferty, Schwarz, and colleagues (1). The CD28 homolog cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) (3) was found to be a higher affinity binding partner for B7-1 and B7-2, as compared to CD28 (4). It was presumed that CTLA-4 would also be a stimulatory receptor; however, the dramatic fatal inflammatory phenotype of CTLA-4 deficient (−/−) mice revealed the critical inhibitory function of CTLA-4 (10, 11). Furthermore, the phenotype of CTLA-4−/− mice demonstrated that T-cell costimulatory receptors could deliver inhibitory, as well as stimulatory, second signals for T-cell responses, and provided the first indication that inhibitory second signals (co-inhibitory) could regulate T-cell tolerance. While the B7-1/B7-2:CD28/CTLA-4 pathway is the best characterized T-cell costimulatory pathway, there are now many additional costimulatory pathways. These pathways fall into two major families: the immunoglobulin (Ig) superfamily, which includes the B7/CD28, CD2, and T-cell Ig and mucin domain (TIM) families, and the tumor necrosis family (TNF)/TNF receptor (TNFR) family. Reviews in this issue of Immunol Rev discuss the functions of pathways within all of these families. These articles not only review the current understanding of costimulation on the fundamental level, but also the therapeutic opportunities and challenges of targeting costimulatory pathways for treating autoimmunity, graft rejection, cancer and infectious diseases. In this introduction, I provide perspectives on key functions of costimulatory pathways, highlighting recent advances, and discuss how this progress is changing our view of the functions of T-cell costimulation.

Novel role for TNF-alpha in memory CD8 T cell-mediated protection against liver stage Plasmodium berghei infection (129.27)

Abstract Malaria results in &amp;gt;1,000,000 deaths per year worldwide. Although no licensed vaccine yet exists, much effort is currently focused on subunit vaccines that elicit CD8 T cell responses directed against liver stage antigens. Multiple T cell effector molecules play a role in anti-microbial immunity mediated by memory CD8 T cells including IFN-gamma, perforin, TRAIL, FasL and TNF-alpha. However, it is not known which effector pathways are required for memory CD8 T cell-mediated immunity to liver stage Plasmodium infection. Here, we used a novel immunization strategy to generate memory CD8 T cells in the wild type and gene-deficient BALB/c mouse model of Plasmodium berghei sporozoite infection to examine the role of these T cell effector molecules in resistance to the liver stage infection. In contrast to previous work examining protection mediated by TCR transgenic effector CD8 T cells, our studies reveal that endogenous memory CD8 T cell-mediated protection is in part IFN-gamma-dependent. Importantly, we also show that neutralization of TNF-alpha in immunized mice markedly reduces memory CD8 T cell-mediated protection. Thus, our studies identify both IFN-gamma and TNF-alpha as important components of the non-cytolytic pathways that underlie memory CD8 T cell-mediated immunity against liver stage Plasmodium infection. Supported by grants from the NIH (to JTH) and an NRSA fellowship (T32AI007260-23 to NSB).

Innovation in immunotherapy

Innovation in immunotherapy

IL‐17/Th17 in anti‐fungal immunity: What's new?

How the immune system tailors protective responses to suit the infectious challenge while limiting damage to the host is an emerging theme in T-cell biology. Although many studies have focused on the pathological aspects of IL-17-producing T cells in many autoimmune diseases, their role in protective anti-microbial immunity has also been increasingly recognized. This increased recognition also applies to their role in anti-fungal immunity; however, the role of IL-17-producing T cells in protection versus pathology in fungal infections is still controversial. Although both positive and negative effects on immune resistance have been attributed to the IL-23/Th17 axis in experimental models of fungal infections, defective Th17 cell differentiation has been linked to recurrent pneumonia by filamentous fungi and the occurrence of mucocutaneous candidiasis in patients with primary immunodeficiencies. Here we discuss how recent findings in experimental candidiasis and aspergillosis shed new lights on the contribution of Th17 cells to resistance and pathology to fungi.

B Cells and Transplantation: An Educational Resource

Allogeneic hematopoietic cell transplantation (HCT) offers a unique opportunity to monitor the development of immune reconstitution using the patient as his or her own control. Furthermore, the donor serves as an HLA-identical normal control and source of adoptive immune transfer. Donor hematopoietic stem cells (HSCs) differentiate and proliferate to repopulate all blood lineages providing normal cell numbers of red blood cells, platelets, and neutrophils. Successful immune reconstitution and protection from infection requires antimicrobial B cell and antibody development. Studies of B cell reconstitution after HCT have primarily examined immunoglobulin concentration, B cell quantification, and antimicrobial antibody development in relation to donor/recipient serologic status or vaccination. Following HCT, humoral immunity has three distinct contributions. First, recipient antibody persists with an average half-life of 30–60 days, and some recipient plasma cells persist for years following allogeneic HCT (1) providing protective antimicrobial humoral immunity (2). Some recipient anti-donor alloimmune responses are detrimental contributring to primary graft rejection (3, 4) and prolonged red cell aplasia when donors and recipients are ABO major mismatched (5, 6). Second, donor grafts contain naive and memory B cells that have already undergone positive and negative selection in the HLA-identical donor and contribute adoptive antimicrobial and alloreactive B cells. Third, B cells reconstituting from donor hematopoietic stem cells (HSC) recognizing disparate recipient antigens as “self”, will be clonally deleted preventing alloreactive responses, but remain capable of responding to infectious challenges and vaccinations. This educational session will consider B cell responses following allogeneic HCT as they contribute to 1) vaccine induced antimicrobial immunity, 2) autoimmune responses, and 3) allogeneic antibody responses. We will discuss a B cell role in chronic GVHD pathogenesis, review anti-B cell chronic GVHD therapy using rituximab, and finally consider the pathogenic role of agonistic antibodies targeting platelet derived growth factor receptor (PDGFR).

Alarmins and antimicrobial immunity

Alarmins are endogenous mediators capable of enhancing innate and adaptive immune response through induction of concomitant recruitment and activation of antigen-presenting cells. Here we provide a brief overview of various alarmins, highlight their critical roles in innate and adaptive antimicrobial immunity, and speculate on potential usage of alarmins in combating aspergillosis.

Potent and Broad-Spectrum Antimicrobial Activity of CXCL14 Suggests an Immediate Role in Skin Infections

The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the chemokine CXCL14 is a broad-spectrum AMP with killing activity for cutaneous gram-positive bacteria and Candida albicans as well as the gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human beta-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity.

Insights into the Role of STAT3 in Human Lymphocyte Differentiation as Revealed by the Hyper-IgE Syndrome

"Experiments of nature" due to single gene mutations resulting in human immunodeficiency states have revealed critical roles for several genes in regulating lymphocyte development and the generation of protective immunity. Recently, heterozygous mutations in STAT3 were found to cause autosomal dominant hyper-IgE syndrome, a condition affecting not only the immune system but also other mesenchymal and ectodermal tissues, including bones, cranium, teeth, and skin. STAT proteins operate to integrate signals from surface receptors, including cytokine receptors, that regulate growth and differentiation of multiple cell lineages. In this article, we will review how the study of STAT3 deficiency in humans and mice has highlighted nonredundant roles of STAT3, and of specific cytokines, in diverse cellular processes such as antimicrobial immunity and protection at epithelial barriers, the generation of functional humoral immune responses, bone formation, and keratinocyte biology.

IgG Allotypes Reveal That Antimicrobial Humoral Immunity Persists after Reduced-Intensity Hematopoietic Cell Transplantation

Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for hematologic malignancies through graft-versus-leukemia/lymphoma (GVL) responses and replacement of the patient's immune system. Conditioning intensity may influence hematopoietic reconstitution, persistent recipient immunity, and donor alloimmune responses. High-dose conditioning (HDC) causes rapid conversion to full donor T- and B-cell chimerism, while patients who undergo reduced-intensity conditioning (RIC) progress through a dynamic mixed chimerism extending weeks to months. We hypothesize that long-lived recipient plasma cells persist, providing beneficial antimicrobial serologic immunity, while donor B cells target allo-antigens, contributing to both GVL and GVHD. Furthermore, recipient humoral immunity persists longer following RIC than HDC, leading to fewer infections and decreased transplant-related mortality.Inherited polymorphisms in IgG heavy chain constant regions can be recognized by allotype-specific monoclonal antibodies and thus distinguish donor and recipient antibodies. We developed a quantitative ELISA by coating mouse monoclonal antibodies specific for the G1m(f) and G2m(n) allotypes. Serial dilutions of 63 patients and their donors were incubated and detected by alkaline phosphatase-conjugated anti-human IgG polyclonal antibody. Twenty-eight HDC patients were conditioned with VP-16 and total body irradiation, while 35 RIC patients received total lymphoid irradiation and anti-thymocyte globulin (TLI-ATG). One hundred fifteen of the total 126 subjects (91%) expressed G1m(f) (null allele frequency: 0.29), and 100 of 126 (79%) expressed G2m(n) (null allele frequency: 0.45). Twenty-six of the 63 pairs (41%) were informative because either the donor or recipient was homozygous null for an allotype. Following both RIC and HDC when the recipient was allotype null, the donor allotype was first detected six months post-HCT and reached 50–80% of donor levels by 12 months (n=8 pairs). Eighteen donors were homozygous allotype null, and recipient-specific antibody was prospectively determined. By 12 months after HDC, recipient antibody levels fell to <10% of pre-HCT allotype levels (n=11 pairs). In contrast, RIC patients retained the allotype at >90% of pre-HCT levels through 12 months post-HCT (n=7 pairs), demonstrating a significant difference from HDC patients in one-year median recipient allotype levels (p=0.016). We confirmed RIC humoral immune persistence by DNA chimerism detection of CD38+CD138+ plasma cells from bone marrow aspirates collected 12 months post-HCT. Analysis revealed 30–58% donor levels of plasma cells, suggesting that RIC patients maintain long-lived plasma cells.Allotype-informative patients were measured by ELISA for total IgG and allotype-specific IgG against Epstein-Barr virus (EBV), varicella zoster virus (VZV), pneumococcus, and influenza. HDC patients displayed loss of recipient antimicrobial allotype between five and eight months in conjunction with loss of bulk allotype. RIC patients maintained >90% pre-HCT allotype levels through one year correlating to levels of total IgG against all four infectious agents when they were seropositive before HCT. In addition, new donorspecific responses to influenza, pneumococcus, and VZV were detected in both sets of patients within seven to 14 months.In the allotype-informative setting, H-Y antibody analysis confirmed that donor-derived allogeneic antibodies against UTY2 and DDX3Y developed after nine and 12 months, respectively, and persisted in both HDC and RIC male patients with female donors.In summary, RIC recipients benefit from a twofold effect: persistent recipient-derived antimicrobial humoral immunity and donor-derived allogeneic B-cell responses. Our studies may explain why RIC results in decreased infectious complications in the post-HCT setting.

Rapid NKT Cell Responses Are Self-Terminating during the Course of Microbial Infection

NKT cells play a protective role in immune responses against infectious pathogens. However, when the NKT cell response to infection is initiated and terminated is unknown. In this study, we demonstrate that NKT cells become activated, proliferate, and exert their effector function before MHC-restricted T cells during infection with Mycobacterium bovis bacillus Calmette-Guérin in mice. After a cell expansion phase, NKT cells underwent cell death, which contracts their numbers back to baseline. Surprisingly, despite ongoing infection, the remaining NKT cells were profoundly unresponsive to TCR stimulation, while MHC-restricted T cells were vigorously proliferating and producing IFN-gamma. Similarly, we show that NKT cells became unresponsive in uninfected mice after receiving a single exposure to a TLR agonist LPS, suggesting that NKT cell unresponsiveness may be a major mechanism of terminating their response in many infectious conditions. This characterization of the NKT cell response in antimicrobial immunity indicates that rapid NKT cell activation contributes to the innate phase of the response to the infectious pathogen, but then, the NKT cell response is shut down by two mechanisms; apoptotic contraction and marked unresponsiveness to TCR stimulation, as a synchronized hand off to MHC-restricted T cells occurs.

Dysregulation of the host mevalonate pathway during early bacterial infection activates human TCR γδ cells

Primates, but not rodents, have T cell receptor Vgamma9-Vdelta2 T cells bridging innate and adaptive antimicrobial immunity. This T cell population is activated by prenyl pyrophosphates isolated from microbial or eukaryotic cells. Although the microbial metabolites are more active than the cellular ones, their involvement in TCR gammadelta activation during infection has not been studied. Here, we show that, during the initial phases of infections with Escherichia coli and Staphylococcus aureus, TCR gammadelta cells are activated by endogenous mevalonate metabolites. Infections with low bacteria inocula up-regulate the production and accumulation of host-derived TCR gammadelta stimulatory antigens within 1 h, which is followed by a peak of TCR gammadelta cell activation at 5 h. Infections induce the accumulation and dephosphorylation of the hydroxymethylglutaryl-coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway, resulting in increased activity of this enzyme and in increased synthesis of intermediate metabolites. Thus, primates have evolved the ability to readily respond to bacterial infection by sensing the dysregulation of the mevalonate pathway within infected cells, as a mechanism of immediate antimicrobial immunity.

Effect of Citrulline and Glutamine on Nitric Oxide Production in RAW 264.7 Cells in an Arginine‐Depleted Environment

Nitric oxide (NO) is a highly reactive free radical essential for antimicrobial and tumor immunity as well as endothelial function. Arginine is a limiting factor in NO synthesis. Citrulline can be converted to arginine and might restore NO production when arginine availability is limited, while glutamine may competitively inhibit citrulline availability. We aimed to assess how these amino acids interact to generate NO using an in vitro model. RAW 264.7 cells were exposed to various amino acid concentrations before and after lipopolysaccharide (LPS) stimulation, and NO production was assessed. NO production directly correlated up to 200 microM with arginine available after LPS stimulation (R(2) = 0.99). Provided the same arginine concentrations following LPS stimulation, low arginine precultured cells produced significantly less NO than high arginine precultured cells (P < .01). Citrulline added to low arginine preculture significantly increased NO production compared to cells in low arginine alone (P < .01). When glutamine was withdrawn before and after LPS stimulation, cells precultured in low arginine and citrulline produced NO equivalent to that of high arginine precultured cells. Additional citrulline provided after LPS stimulation additionally improved NO production beyond that observed in cells precultured in high arginine (P < .01), and NO production became less dependent on arginine availability (R(2) = 0.78). Arginine availability is a limiting factor for NO production. Citrulline is a potential substitute to restore NO production when arginine availability is limited. Glutamine appears to be an important modulator that interferes with citrulline-mediated NO production.

Murine Bactericidal/Permeability-Increasing Protein Inhibits the Endotoxic Activity of Lipopolysaccharide and Gram-Negative Bacteria

Recognition of LPS by TLR4 initiates inflammatory responses inducing potent antimicrobial immunity. However, uncontrolled inflammatory responses can be detrimental. To prevent the development of septic shock during an infection with Gram-negative bacteria, the immune system has developed mechanisms to neutralize LPS by specialized proteins. In this study, we report the recombinant expression and functional characterization of the mouse homolog of human bactericidal/permeability-increasing protein (BPI). Purified recombinant mouse BPI was able to neutralize LPS-mediated activation of macrophages and to block LPS-dependent maturation of dendritic cells. Recombinant mouse BPI neutralized the capacity of Gram-negative bacteria to activate immune cells, but did not influence the stimulatory properties of Gram-positive bacteria. Unlike human BPI, mouse BPI failed to kill or inhibit the growth of Pseudomonas aeruginosa. Together, these data demonstrate that murine BPI is a potent LPS-neutralizing protein that may limit innate immune responses during Gram-negative infections.

PD-1 and Its Ligands in Tolerance and Immunity

Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, deliver inhibitory signals that regulate the balance between T cell activation, tolerance, and immunopathology. Immune responses to foreign and self-antigens require specific and balanced responses to clear pathogens and tumors and yet maintain tolerance. Induction and maintenance of T cell tolerance requires PD-1, and its ligand PD-L1 on nonhematopoietic cells can limit effector T cell responses and protect tissues from immune-mediated tissue damage. The PD-1:PD-L pathway also has been usurped by microorganisms and tumors to attenuate antimicrobial or tumor immunity and facilitate chronic infection and tumor survival. The identification of B7-1 as an additional binding partner for PD-L1, together with the discovery of an inhibitory bidirectional interaction between PD-L1 and B7-1, reveals new ways the B7:CD28 family regulates T cell activation and tolerance. In this review, we discuss current understanding of the immunoregulatory functions of PD-1 and its ligands and their therapeutic potential.

Surface antigens are rapidly separated from bacterium‐sized microspheres in the subcapsular sinus and acquired by antigen‐specific follicular B cells

Antibody production is critical for antimicrobial immunity. The initial step in this process is the binding of antigen to the B cell receptor. Yet, the size of many microbes prevents them from entering the lymph node follicles where naïve B cells reside. Here, we show that antigen‐specific follicular B cells rapidly acquired antigen, produced peptide:MHC II complexes, and secreted antibody in a T cell‐dependent fashion following subcutaneous injection of bacterium‐sized antigen‐coupled particles even though the particles were confined to the subcapsular sinus. This form of B cell antigen acquisition did not depend on dendritic cells or subcapsular sinus macrophages. These results suggest that proteins released from the surface of microbes directly access the follicles to initiate the humoral response.

Inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein

TNFalpha is an important cytokine in antimicrobial immunity and inflammation. The receptor-interacting protein RIP1 is an essential component of the TNF receptor 1 signaling pathway that mediates the activation of NF-kappaB, MAPKs, and programmed cell death. It also transduces signals derived from Toll-like receptors and intracellular sensors of DNA damage and double-stranded RNA. Here, we show that the murine CMV M45 protein binds to RIP1 and inhibits TNFalpha-induced activation of NF-kappaB, p38 MAPK, and caspase-independent cell death. M45 also inhibited NF-kappaB activation upon stimulation of Toll-like receptor 3 and ubiquitination of RIP1, which is required for NF-kappaB activation. Hence, M45 functions as a viral inhibitor of RIP1-mediated signaling. The results presented here reveal a mechanism of viral immune subversion and demonstrate how a viral protein can simultaneously block proinflammatory and innate immune signaling pathways by interacting with a central mediator molecule.

The PD-1/PD-L costimulatory pathway critically affects host resistance to the pathogenic fungusHistoplasma capsulatum

The PD-1 costimulatory receptor inhibits T cell receptor signaling upon interacting with its ligands PD-L1 and PD-L2. The PD-1/PD-L pathway is critical in maintaining self-tolerance. In this study, we examined the role of PD-1 in a mouse model of acute infection with Histoplasma capsulatum, a major human pathogenic fungus. In a lethal model of histoplasmosis, all PD-1-deficient mice survived infection, whereas the wild-type mice died with disseminated disease. PD-L expression on macrophages and splenocytes was up-regulated during infection, and macrophages from infected mice inhibited in vitro T cell activation. Of interest, antibody blocking of PD-1 significantly increased survival of lethally infected wild-type mice. Thus, our studies extend the role of the PD-1/PD-L pathway in regulating antimicrobial immunity to fungal pathogens. The results show that the PD-1/PD-L pathway has a key role in the regulation of antifungal immunity, and suggest that manipulation of this pathway represents a strategy of immunotherapy for histoplasmosis.