Monoclonal antibodies derived from B cells in subjects with cystic fibrosis reduce Pseudomonas aeruginosa burden in mice.

Pseudomonas aeruginosa (PA) is an opportunistic, frequently multidrug-resistant pathogen that can cause severe infections in hospitalized patients. Antibodies against the PA virulence factor, PcrV, protect from death and disease in a variety of animal models. However, clinical trials of PcrV-binding antibody-based products have thus far failed to demonstrate benefit. Prior candidates were derivations of antibodies identified using protein-immunized animal systems and required extensive engineering to optimize binding and/or reduce immunogenicity. Of note, PA infections are common in people with cystic fibrosis (pwCF), who are generally believed to mount normal adaptive immune responses. Here we utilized a tetramer reagent to detect and isolate PcrV-specific B cells in pwCF and, via single-cell sorting and paired-chain sequencing, identified the B cell receptor (BCR) variable region sequences that confer PcrV-specificity. We derived multiple high affinity anti-PcrV monoclonal antibodies (mAbs) from PcrV-specific B cells across 3 donors, including mAbs that exhibit potent anti-PA activity in a murine pneumonia model. This robust strategy for mAb discovery expands what is known about PA-specific B cells in pwCF and yields novel mAbs with potential for future clinical use.

Plasmodium infection disrupts the T follicular helper cell response to heterologous immunization.

Naturally acquired immunity to malaria develops only after many years and repeated exposures, raising the question of whether Plasmodium parasites, the etiological agents of malaria, suppress the ability of dendritic cells (DCs) to activate optimal T cell responses. We demonstrated recently that B cells, rather than DCs, are the principal activators of CD4+ T cells in murine malaria. In the present study, we further investigated factors that might prevent DCs from priming Plasmodium-specific T helper cell responses. We found that DCs were significantly less efficient at taking up infected red blood cells (iRBCs) compared to soluble antigen, whereas B cells more readily bound iRBCs. To assess whether DCs retained the capacity to present soluble antigen during malaria, we measured responses to a heterologous protein immunization administered to naïve mice or mice infected with P. chabaudi. Antigen uptake, DC activation, and expansion of immunogen-specific T cells were intact in infected mice, indicating DCs remained functional. However, polarization of the immunogen-specific response was dramatically altered, with a near-complete loss of germinal center T follicular helper cells specific for the immunogen, accompanied by significant reductions in antigen-specific B cells and antibody. Our results indicate that DCs remain competent to activate T cells during Plasmodium infection, but that T cell polarization and humoral responses are severely disrupted. This study provides mechanistic insight into the development of both Plasmodium-specific and heterologous adaptive responses in hosts with malaria.

Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict Plasmodium parasites.

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum-specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.

Gasdermin-D and Caspase-7 are the key Caspase-1/8 substrates downstream of the NAIP5/NLRC4 inflammasome required for restriction of Legionella pneumophila.

Inflammasomes are cytosolic multi-protein complexes that detect infection or cellular damage and activate the Caspase-1 (CASP1) protease. The NAIP5/NLRC4 inflammasome detects bacterial flagellin and is essential for resistance to the flagellated intracellular bacterium Legionella pneumophila. The effectors required downstream of NAIP5/NLRC4 to restrict bacterial replication remain unclear. Upon NAIP5/NLRC4 activation, CASP1 cleaves and activates the pore-forming protein Gasdermin-D (GSDMD) and the effector caspase-7 (CASP7). However, Casp1-/- (and Casp1/11-/-) mice are only partially susceptible to L. pneumophila and do not phenocopy Nlrc4-/-mice, because NAIP5/NLRC4 also activates CASP8 for restriction of L. pneumophila infection. Here we show that CASP8 promotes the activation of CASP7 and that Casp7/1/11-/- and Casp8/1/11-/- mice recapitulate the full susceptibility of Nlrc4-/- mice. Gsdmd-/- mice exhibit only mild susceptibility to L. pneumophila, but Gsdmd-/-Casp7-/- mice are as susceptible as the Nlrc4-/- mice. These results demonstrate that GSDMD and CASP7 are the key substrates downstream of NAIP5/NLRC4/CASP1/8 required for resistance to L. pneumophila.

FCRL5+ Memory B Cells Exhibit Robust Recall Responses.

FCRL5+ atypical memory B cells (atMBCs) expand in many chronic human infections, including recurrent malaria, but studies have drawn opposing conclusions about their function. Here, in mice infected with Plasmodium chabaudi, we demonstrate expansion of an antigen-specific FCRL5+ population that is distinct from previously described FCRL5+ innate-like murine subsets. Comparative analyses reveal overlapping phenotypic and transcriptomic signatures between FCRL5+ B cells from Plasmodium-infected mice and atMBCs from Plasmodium-exposed humans. In infected mice, FCRL5 is expressed on the majority of antigen-specific germinal-center-derived memory B cells (MBCs). Upon challenge, FCRL5+ MBCs rapidly give rise to antibody-producing cells expressing additional atypical markers, demonstrating functionality in vivo. Moreover, atypical markers are expressed on antigen-specific MBCs generated by immunization in both mice and humans, indicating that the atypical phenotype is not restricted to chronic settings. This study resolves conflicting interpretations of atMBC function and suggests FCRL5+ B cells as an attractive target for vaccine strategies.

Alternate splicing of transcripts upon Mycobacterium tuberculosis infection impacts the expression of functional protein domains.

Previously, we reported that infection of human macrophages with Mycobacterium tuberculosis (Mtb) results in massive alterations in the pattern of RNA splicing in the host. The finding gained significance since alternate spliced variants of a same gene may have substantially different structure, function, stability, interaction partners, localization, and so forth, owing to inclusion or exclusion of specific exons. To establish a proof-of-concept; on how infection-induced RNA splicing could impact protein functions, here we used RNA-seq data from THP-1 macrophages that were infected with clinical isolate of Mtb. In addition to re-establishing the fact that Mtb infection may cause strain specific alterations in RNA splicing, we also developed a new analysis pipeline resulting in characterization of domain maps of the transcriptome post-infection. For the sake of simplicity, we restricted our analysis to all the kinases in the human genome and considered only pfam classified protein domains and checked their frequency of inclusion or exclusion due to alternate splicing across the conditions and time points. We report massive alterations in the domain architecture of most regulated proteins across the entire kinases highlighting the physiological importance of such an understanding. This study paves way for more detailed analysis of different functional classes of proteins and perturbations to their domain architecture as a consequence of mycobacterial infections. Such analysis would yield unprecedented depth to our understanding of host-pathogen interaction and allow in a more systematic manner targeting of host pathways for controlling the infections. © 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(9):845-854, 2018.

Detection of Succinate by Intestinal Tuft Cells Triggers a Type 2 Innate Immune Circuit.

In the small intestine, type 2 responses are regulated by a signaling circuit that involves tuft cells and group 2 innate lymphoid cells (ILC2s). Here, we identified the microbial metabolite succinate as an activating ligand for small intestinal (SI) tuft cells. Sequencing analyses of tuft cells isolated from the small intestine, gall bladder, colon, thymus, and trachea revealed that expression of tuft cell chemosensory receptors is tissue specific. SI tuft cells expressed the succinate receptor (SUCNR1), and providing succinate in drinking water was sufficient to induce a multifaceted type 2 immune response via the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis and a tritrichomonad protist both secreted succinate as a metabolite. In vivo sensing of the tritrichomonad required SUCNR1, whereas N. brasiliensis was SUCNR1 independent. These findings define a paradigm wherein tuft cells monitor microbial metabolites to initiate type 2 immunity and suggest the existence of other sensing pathways triggering the response to helminths.

NKTeeing Up B Cell Responses to Viral Infection.

Activated B cells mature in germinal centers (GCs), but GC initiation during infection is poorly understood. Gaya et al. (2018) show that NKT cells, activated by CD169+ macrophages, produce an early wave of interleukin-4 (IL-4) that promotes GC formation during viral infection.

Correction: Alternate splicing of transcripts shape macrophage response to Mycobacterium tuberculosis infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006236.].

Alternate splicing of transcripts shape macrophage response to Mycobacterium tuberculosis infection.

Transcriptional reprogramming of macrophages upon Mycobacterium tuberculosis (Mtb) infection is widely studied; however, the significance of alternate splicing (AS) in shaping cellular responses to mycobacterial infections is not yet appreciated. Alternate splicing can influence transcript stability or structure, function and localization of corresponding proteins thereby altering protein stoichiometry and physiological consequences. Using comprehensive analysis of a time-series RNA-seq data obtained from human macrophages infected with virulent or avirulent strains of Mtb, we show extensive remodeling of alternate splicing in macrophage transcriptome. The global nature of this regulation was evident since genes belonging to functional classes like trafficking, immune response, autophagy, redox and metabolism showed marked departure in the pattern of splicing in the infected macrophages. The systemic perturbation of splicing machinery in the infected macrophages was apparent as genes involved at different stages of spliceosome assembly were also regulated at the splicing level. Curiously there was a considerable increase in the expression of truncated/non-translatable variants of several genes, specifically upon virulent infections. Increased expression of truncated transcripts correlated with a decline in the corresponding protein levels. We verified the physiological relevance for one such candidate gene RAB8B; whose truncated variant gets enriched in H37Rv infected cells. Upon tweaking relative abundance of longer or shorter variants of RAB8B transcripts by specialized transduction, mycobacterial targeting to lysosomes could be promoted or blocked respectively, which also resulted in corresponding changes in the bacterial survival. Our results show RAB8B recruitment to the mycobacterial phagosomes is required for phagosome maturation. Thus the abundance of truncated RAB8B variant helps virulent Mtb survival by limiting the RAB8B levels in the cells, a mechanism which we subsequently verified in human primary macrophages. Taken together we demonstrate alternate splicing as a new locus of intervention by Mtb and provide attractive alternative to exploit for novel drug targets against Mtb.

Correction: Macrophage Colony Stimulating Factor Derived from CD4+ T Cells Contributes to Control of a Blood-Borne Infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006046.].

Macrophage Colony Stimulating Factor Derived from CD4+ T Cells Contributes to Control of a Blood-Borne Infection.

Dynamic regulation of leukocyte population size and activation state is crucial for an effective immune response. In malaria, Plasmodium parasites elicit robust host expansion of macrophages and monocytes, but the underlying mechanisms remain unclear. Here we show that myeloid expansion during P. chabaudi infection is dependent upon both CD4+ T cells and the cytokine Macrophage Colony Stimulating Factor (MCSF). Single-cell RNA-Seq analysis on antigen-experienced T cells revealed robust expression of Csf1, the gene encoding MCSF, in a sub-population of CD4+ T cells with distinct transcriptional and surface phenotypes. Selective deletion of Csf1 in CD4+ cells during P. chabaudi infection diminished proliferation and activation of certain myeloid subsets, most notably lymph node-resident CD169+ macrophages, and resulted in increased parasite burden and impaired recovery of infected mice. Depletion of CD169+ macrophages during infection also led to increased parasitemia and significant host mortality, confirming a previously unappreciated role for these cells in control of P. chabaudi. This work establishes the CD4+ T cell as a physiologically relevant source of MCSF in vivo; probes the complexity of the CD4+ T cell response during type 1 infection; and delineates a novel mechanism by which T helper cells regulate myeloid cells to limit growth of a blood-borne intracellular pathogen.

A Novel Model of Asymptomatic Plasmodium Parasitemia That Recapitulates Elements of the Human Immune Response to Chronic Infection.

In humans, immunity to Plasmodium sp. generally takes the form of protection from symptomatic malaria (i.e., 'clinical immunity') rather than infection ('sterilizing immunity'). In contrast, mice infected with Plasmodium develop sterilizing immunity, hindering progress in understanding the mechanistic basis of clinical immunity. Here we present a novel model in which mice persistently infected with P. chabaudi exhibit limited clinical symptoms despite sustaining patent parasite burdens for many months. Characterization of immune responses in persistently infected mice revealed development of CD4+ T cell exhaustion, increased production of IL-10, and expansion of B cells with an atypical surface phenotype. Additionally, persistently infected mice displayed a dramatic increase in circulating nonclassical monocytes, a phenomenon that we also observed in humans with both chronic Plasmodium exposure and asymptomatic infection. Following pharmacological clearance of infection, previously persistently infected mice could not control a secondary challenge, indicating that persistent infection disrupts the sterilizing immunity that typically develops in mouse models of acute infection. This study establishes an animal model of asymptomatic, persistent Plasmodium infection that recapitulates several central aspects of the immune response in chronically exposed humans. As such, it provides a novel tool for dissection of immune responses that may prevent development of sterilizing immunity and limit pathology during infection.

FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure.

Exposure to Plasmodium falciparum is associated with circulating "atypical" memory B cells (atMBCs), which appear similar to dysfunctional B cells found in HIV-infected individuals. Functional analysis of atMBCs has been limited, with one report suggesting these cells are not dysfunctional but produce protective antibodies. To better understand the function of malaria-associated atMBCs, we performed global transcriptome analysis of these cells, obtained from individuals living in an area of high malaria endemicity in Uganda. Comparison of gene expression data suggested down-modulation of B cell receptor signaling and apoptosis in atMBCs compared to classical MBCs. Additionally, in contrast to previous reports, we found upregulation of Fc receptor-like 5 (FCRL5), but not FCRL4, on atMBCs. Atypical MBCs were poor spontaneous producers of antibody ex vivo, and higher surface expression of FCRL5 defined a distinct subset of atMBCs compromised in its ability to produce antibody upon stimulation. Moreover, higher levels of P. falciparum exposure were associated with increased frequencies of FCRL5+ atMBCs. Together, our findings suggest that FCLR5+ identifies a functionally distinct, and perhaps dysfunctional, subset of MBCs in individuals exposed to P. falciparum.

JUNB is a key transcriptional modulator of macrophage activation.

Activated macrophages are crucial for restriction of microbial infection but may also promote inflammatory pathology in a wide range of both infectious and sterile conditions. The pathways that regulate macrophage activation are therefore of great interest. Recent studies in silico have putatively identified key transcription factors that may control macrophage activation, but experimental validation is lacking. In this study, we generated a macrophage regulatory network from publicly available microarray data, employing steps to enrich for physiologically relevant interactions. Our analysis predicted a novel relationship between the AP-1 family transcription factor Junb and the gene Il1b, encoding the pyrogen IL-1ß, which macrophages express upon activation by inflammatory stimuli. Previously, Junb has been characterized primarily as a negative regulator of the cell cycle, whereas AP-1 activity in myeloid inflammatory responses has largely been attributed to c-Jun. We confirmed experimentally that Junb is required for full expression of Il1b, and of additional genes involved in classical inflammation, in macrophages treated with LPS and other immunostimulatory molecules. Furthermore, Junb modulates expression of canonical markers of alternative activation in macrophages treated with IL-4. Our results demonstrate that JUNB is a significant modulator of both classical and alternative macrophage activation. Further, this finding provides experimental validation for our network modeling approach, which will facilitate the future use of gene expression data from open databases to reveal novel, physiologically relevant regulatory relationships.

Loss of Toll-like receptor 7 alters cytokine production and protects against experimental cerebral malaria.

BACKGROUND: Malaria, caused by Plasmodium sp. parasites, is a leading cause of global morbidity and mortality. Cerebral malaria, characterized by neurological symptoms, is a life-threatening complication of malaria affecting over 500,000 young children in Africa every year. Because of the prevalence and severity of cerebral malaria, a better understanding of the underlying molecular mechanisms of its pathology is desirable and could inform future development of therapeutics. This study sought to clarify the role of Toll-like receptors (TLRs) in promoting immunopathology associated with cerebral malaria, with a particular focus on the understudied TLR7. METHODS: Using the Plasmodium berghei ANKA mouse model of experimental cerebral malaria, C57BL/6 mice deficient in various TLRs were infected, and their resistance to cerebral malaria and immune activation through cytokine production were measured. RESULTS: Loss of TLR7 conferred partial protection against fatal experimental cerebral malaria. Additionally, loss of TLR signalling dysregulated the cytokine profile, resulting in a shift in the cytokine balance towards those with more anti-inflammatory properties. CONCLUSION: This work identifies signalling through TLR7 as a source of pathology in experimental cerebral malaria.

Toll-like receptor 7 mediates early innate immune responses to malaria.

Innate immune recognition of malaria parasites is the critical first step in the development of the host response. At present, Toll-like receptor 9 (TLR9) is thought to play a central role in sensing malaria infection. However, we and others have observed that Tlr9(-/-) mice, in contrast to mice deficient in the downstream adaptor, Myeloid differentiation primary response gene 88 (MYD88), exhibit few deficiencies in immune function during early infection with the malaria parasite Plasmodium chabaudi, implying that another MYD88-dependent receptor also contributes to the antimalarial response. Here we use candidate-based screening to identify TLR7 as a key sensor of early P. chabaudi infection. We show that TLR7 mediates a rapid systemic response to infection through induction of cytokines such as type I interferons (IFN-I), interleukin 12, and gamma interferon. TLR7 is also required for induction of IFN-I by other species and strains of Plasmodium, including an etiological agent of human disease, P. falciparum, suggesting that malaria parasites harbor a common pathogen-associated molecular pattern (PAMP) recognized by TLR7. In contrast to the nonredundant requirement for TLR7 in early immune activation, sensing through both TLR7 and TLR9 was required for proinflammatory cytokine production and immune cell activation during the peak of parasitemia. Our findings indicate that TLR7 plays a central role in early immune activation during malaria infection, whereas TLR7 and TLR9 contribute combinatorially to immune responses as infection progresses.

IL-1α signaling initiates the inflammatory response to virulent Legionella pneumophila in vivo.

Legionella pneumophila is an intracellular bacterial pathogen that is the cause of a severe pneumonia in humans called Legionnaires' disease. A key feature of L. pneumophila pathogenesis is the rapid influx of neutrophils into the lungs, which occurs in response to signaling via the IL-1R. Two distinct cytokines, IL-1α and IL-1ß, can stimulate the type I IL-1R. IL-1ß is produced upon activation of cytosolic sensors called inflammasomes that detect L. pneumophila in vitro and in vivo. Surprisingly, we find no essential role for IL-1ß in neutrophil recruitment to the lungs in response to L. pneumophila. Instead, we show that IL-1α is a critical initiator of neutrophil recruitment to the lungs of L. pneumophila-infected mice. We find that neutrophil recruitment in response to virulent L. pneumophila requires the production of IL-1α specifically by hematopoietic cells. In contrast to IL-1ß, the innate signaling pathways that lead to the production of IL-1α in response to L. pneumophila remain poorly defined. In particular, although we confirm a role for inflammasomes for initiation of IL-1ß signaling in vivo, we find no essential role for inflammasomes in production of IL-1α. Instead, we propose that a novel host pathway, perhaps involving inhibition of host protein synthesis, is responsible for IL-1α production in response to virulent L. pneumophila. Our results establish IL-1α as a critical initiator of the inflammatory response to L. pneumophila in vivo and point to an important role for IL-1α in providing an alternative to inflammasome-mediated immune responses in vivo.

Caspase-11 protects against bacteria that escape the vacuole.

Caspases are either apoptotic or inflammatory. Among inflammatory caspases, caspase-1 and -11 trigger pyroptosis, a form of programmed cell death. Whereas both can be detrimental in inflammatory disease, only caspase-1 has an established protective role during infection. Here, we report that caspase-11 is required for innate immunity to cytosolic, but not vacuolar, bacteria. Although Salmonella typhimurium and Legionella pneumophila normally reside in the vacuole, specific mutants (sifA and sdhA, respectively) aberrantly enter the cytosol. These mutants triggered caspase-11, which enhanced clearance of S. typhimurium sifA in vivo. This response did not require NLRP3, NLRC4, or ASC inflammasome pathways. Burkholderia species that naturally invade the cytosol also triggered caspase-11, which protected mice from lethal challenge with B. thailandensis and B. pseudomallei. Thus, caspase-11 is critical for surviving exposure to ubiquitous environmental pathogens.

Activation of host mitogen-activated protein kinases by secreted Legionella pneumophila effectors that inhibit host protein translation.

Studies of innate immunity in metazoans have largely focused on detection of microbial molecules by host pattern recognition receptors (PRRs). A complementary mode of innate immune recognition, based on detection of pathogen-encoded activities, has long been recognized in plants, where it is termed effector-triggered immunity; however, little is known about the possibility of effector-triggered immunity in metazoans. Legionella pneumophila is an intracellular bacterial pathogen that causes Legionnaires' disease, an inflammatory pneumonia. We recently demonstrated that macrophages infected with L. pneumophila exhibit mitogen-activated protein (MAP) kinase (MAPK) activation that is independent of known PRRs but dependent on a functional bacterial secretion system. Here, we show that five secreted L. pneumophila effectors are responsible for the activation of host MAP kinases. These five effectors inhibit host translation, and their activity is required for host MAPK activation. We demonstrate that MAPK activation by these effectors shapes the host transcriptional response to L. pneumophila. Furthermore, we find that uninfected macrophages treated with two different translation inhibitors exhibit activation of MAP kinases and upregulation of target genes, indicating that translation inhibition alone is sufficient to elicit this response in macrophages. MAP kinase pathways are crucial in many aspects of the immune response, including inflammation and cell motility. Our results demonstrate that this important host pathway can be activated in response to a pathogen-encoded activity, adding to an emerging body of evidence in support of this novel mode of innate immune detection in metazoans.