Pathogen-associated Molecular Pattern Recognition Receptors Research Articles

Nodule formation in Bombyx mori larvae is regulated by BmToll10-3

Nodule formation is a two-step cell-mediated immune response that is elicited by the cytokine spätzle1. Spätzle1 is activated within 30 s of invasion by microorganisms via an extracellular signaling pathway that consists of pathogen-associated molecular pattern recognition receptors, C-type lectins, and serine proteases. Here, we investigated a hemocyte molecule that is involved in eliciting the first step of nodule formation. BmToll10-3 was one of 14 Toll homologs identified in the silkworm Bombyx mori; it is an ortholog of Spodoptera exigua Toll. Previous research suggested that SeToll elicits nodule formation, but no evidence was presented to indicate whether SeToll elicited the first or second step of nodule formation. Reverse transcription-polymerase chain reaction and immunostaining confirmed that BmToll10-3 is expressed in granulocytes. To determine whether BmToll10-3 is involved in eliciting the first step of nodule formation, we tested an antiserum raised against BmToll10-3 in a nodule formation assay. The antiserum strongly inhibited the first step of nodule formation in B. mori larvae. Next, we tried to knock out BmToll10-3 using genome editing. Strains that were heterozygous for a truncated BmToll10-3 allele were generated, but no strain that was homozygous for truncated BmToll10-3 was generated. Nonetheless, several healthy homozygous larvae were identified before pupation, and we used these larvae in a nodule formation assay. The larvae that were homozygous for truncated BmToll10-3 did not form nodules. These results suggest that BmToll10-3 is involved in a cellular immunity, nodule formation.

Toll-like receptor 10 has a role in human macrophage response against Streptococcus pneumoniae.

Toll-like receptors (TLRs) are evolutionarily conserved pathogen-associated molecular pattern recognition receptors, and play a critical role in early response against invading pathogens. Even though TLRs have been widely studied, very little is known about the expression and function of TLR10. Till date, neither any data are available on expression of TLR10 in human lungs nor there is any information on function of TLR10 in macrophages. Streptococcus pneumoniae are Gram-positive, alpha-hemolytic, and major causative agent of pneumonia, ear infections, sinus infections, and meningitis. We examined the role of TLR10 in innate immune response to S. pneumoniae infection in U937 cell line-derived human macrophages. We found a significant increase in TLR10 mRNA and protein expression in S. pneumoniae challenged macrophages. TLR10 knockdown resulted in significant reduction of IL-1β, IL-8, IL-17, and TNF-α but not IL-10 expression in infected macrophages. TLR10 knockdown in macrophages reduced nuclear translocation of NF-κB during S. pneumoniae challenge but did not affect the phagocytosis of the bacteria. Taken together, we report the first data on TLR10's role in macrophage response against S. pneumoniae.

Sleep and Microbes.

Sleep is profoundly altered during the course of infectious diseases. The typical response to infection includes an initial increase in nonrapid eye movement sleep (NREMS) followed by an inhibition in NREMS. REMS is inhibited during infections. Bacterial cell wall components, such as peptidoglycan and lipopolysaccharide, macrophage digests of these components, such as muramyl peptides, and viral products, such as viral double-stranded RNA, trigger sleep responses. They do so via pathogen-associated molecular pattern recognition receptors that, in turn, enhance cytokine production. Altered sleep and associated sleep-facilitated fever responses are likely adaptive responses to infection. Normal sleep in physiological conditions may also be influenced by gut microbes because the microbiota is affected by circadian rhythms, stressors, diet, and exercise. Furthermore, sleep loss enhances translocation of viable bacteria from the intestine, which provides another means by which sleep-microbe interactions impact neurobiology.

Abstract B159: Modulation of checkpoint expression in tumor microenvironment by intratumoral administration of a novel TLR9 agonist: Rationale for combination therapy

Abstract Recent advances in the understanding of immune tolerance has led to the targeting of various checkpoints for the treatment of cancer. Several independent clinical studies have pointed out a strong correlation between the clinical activity of checkpoint inhibitors and tumor checkpoint expression. These findings highlight an opportunity to improve cancer immunotherapy by combining checkpoint inhibitors with an immunostimulatory agent that modulates the tumor microenvironment. Our objective was to modulate the tumor microenvironment by activating Toll-like receptor (TLR) 9, a pathogen-associated molecular pattern recognition receptor. In preclinical studies, administration of IMO-2125, a novel TLR9 agonist, led to maturation of dendritic cells and induction of IFN-alpha. We hypothesized that immune activation by a TLR9 agonist administered intratumorally (i.t.) will lead to modulation of immune checkpoints in the tumor microenvironment, allowing these tumors to be targeted with a checkpoint inhibitor. To evaluate the potential of i.t. IMO-2125 therapy and its effect on checkpoint gene expression, we employed three different tumor models: CT26 colon carcinoma, A20 lymphoma, and B16 melanoma mouse models. IMO-2125 was administered at a dose of 50 mcg per treatment, either as a single dose or as multiple doses into a targeted tumor nodule. Blood samples were collected post-dosing for analysis of the cytokine profile. Anti-tumor activity was evaluated by measuring the growth of the injected tumor as well as the distant tumor. Tumor nodules were homogenized to analyze gene expression of selected checkpoints by qPCR. Results showed that a single i.t. injection of IMO-2125 in a CT26 colon carcinoma model induced systemic Th1 type cytokines, and resulted in tumor growth inhibition. Two days post IMO-2125 administration, analysis of gene expression of the treated tumor showed upregulation of IDO1 (13 fold) and PDL1 (2.7 fold), and no changes in expression of PD1 and OX40. Increased IDO1 gene expression correlated with increased IDO protein levels in the treated tumor nodules. In contrast, subcutaneous administration of IMO-2125 showed no substantial anti-tumor effect and no significant changes in checkpoint gene expression despite an induction of similar levels of systemic cytokines as the i.t. treatment. Multiple i.t. injections of IMO-2125 led to inhibition of tumor growth of the injected tumor and the distant tumor in all three tumor models. Tumor growth was correlated with increased CD3+ cells in treated as well as distant tumors. Analysis of the checkpoint gene expression at treated and distant tumor sites was conducted at 1-2 weeks post treatment. There was a significant upregulation of IDO1 and PDL1 gene expression in the treated tumors as well as in distant tumors. In addition to IDO1 and PDL1, depending on the tumor model, other immune checkpoints, including PD1, TIM3, BTLA, LAG3, OX40, and CTLA4, were also modulated. In conclusion, IMO-2125 i.t. treatment led to modulation of the tumor microenvironment by inducing local and systemic Th1 type immune responses, which impacted the levels of checkpoint gene expression. Increased expression of some of these checkpoints may allow tumors to be targeted broadly by checkpoint inhibitor therapy. Citation Format: Wayne Jiang, Daqing Wang, Fugang Zhu, Lakshmi Bhagat, Jillian DiMuzio, Sudhir Agrawal. Modulation of checkpoint expression in tumor microenvironment by intratumoral administration of a novel TLR9 agonist: Rationale for combination therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B159.

Inflammation in acute and chronic pancreatitis.

This report reviews recent animal model and human studies associated with inflammatory responses in acute and chronic pancreatitis. Animal model and limited human acute and chronic pancreatitis studies unravel the dynamic nature of the inflammatory processes and the ability of the immune cells to sense danger and environmental signals. In acute pancreatitis, such molecules include pathogen-associated molecular pattern recognition receptors such as toll-like receptors, and the more recently appreciated damage-associated molecular pattern molecules or 'alarmin' high mobility group box 1 and IL-33. In chronic pancreatitis, a recent understanding of a critical role for macrophage-pancreatic stellate cell interaction offers a potential targetable pathway that can alter fibrogenesis. Microbiome research in pancreatitis is a new field gaining interest but will require further investigation. Immune cell contribution to the pathogenesis of acute and chronic pancreatitis is gaining more appreciation and further understanding in immune signaling presents potential therapeutic targets that can alter disease progression.

IMO-8400, an Antagonist of Toll-like Receptors 7, 8, and 9, in Development for Genetically Defined B-Cell Lymphomas: Safety and Activity in Phase 1 and Phase 2 Clinical Trials

Background. Toll-like receptors (TLRs) are pathogen-associated molecular pattern recognition receptors of the innate immune system. TLRs 7 and 9, which are expressed in human B-cells, respond to DNA- and RNA-based ligands by initiating a signaling cascade mediated through NF-κB, IRAK1/4, BTK, and JAK/STAT. DNA- and RNA-based ligands for TLRs 7 and 9 also are generated as damage-associated molecular patterns in certain autoimmune diseases and malignancies. MYD88 is a key adaptor molecule in TLR signaling. Recently, the oncogenic mutation MYD88 L265P mutation has been described and reported to occur in patients with activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), Waldenström’s macroglobulinemia (WM), and other B-cell malignancies. MYD88 L265P drives over-expression of TLR 7 and 9 signaling, creating an anti-apoptotic microenvironment that promotes cancer cell survival and proliferation. Recent epidemiological data confirm a strong association between the presence of MYD88 L265P and poor outcomes in patients with DLBCL. Specific targeting of signaling initiated by TLRs 7 and 9 therefore is a novel approach to the treatment of B-cell lymphomas harboring the MYD88 L265P mutation. Preclinical data presented at AACR 2014, including from xenograft tumor studies, show that IMO-8400 inhibits cell signaling and reduces tumor growth in WM and DLBCL models harboring the MYD88 L265P mutation. Clinical data from 2 completed trials of IMO-8400 are presented here.Clinical Trial Designs. In a first-in-man Phase 1 trial in healthy subjects, IMO-8400 was administered by s.c. injection at single dosages of 0.1, 0.3, and 0.6 mg/kg and for four weeks at 0.3 and 0.6 mg/kg/week (each n=6). Goals of the Phase 1 trial included safety and tolerability evaluation through single- and multiple-dose escalation, and assessment of TLR engagement. Subsequently, a randomized, placebo-controlled Phase 2 trial was conducted with IMO-8400 in patients with moderate to severe plaque psoriasis at dosages of 0.075, 0.15, 0.3, and 0.6 mg/kg/week for 12 weeks (n=8 or 9 per dosage). Goals of the Phase 2 trial included safety and tolerability assessment over a 12-week treatment period, and evaluation of clinical proof of concept in a disease that involves TLR7 and 9 over-activation.Clinical Trial Results. IMO-8400 was well tolerated across all dose regimens in both studies, with no treatment-related severe or serious adverse events, no drug-related treatment discontinuations, and no pattern of systemic adverse events or laboratory changes. The only treatment-related adverse events in more than one subject were injection site reactions, comprised of generally mild erythema with occasional induration, pruritus, tenderness or pain. In neither trial were there adverse or consistent shifts in laboratory parameters. Specifically, any changes in white or red blood cell parameters, platelet numbers, and liver enzymes or other serum chemistry parameters were similar following IMO-8400 treatment or after placebo treatment. In the Phase 2 trial, clinical proof of mechanism was demonstrated by increased frequency of improvements in psoriasis disease activity in IMO-8400-treated patients across multiple dose levels relative to placebo-treated patients.Conclusion. Antagonism of TLRs 7 and 9 is a novel, scientifically-driven approach to the treatment of B-cell malignancies characterized by presence of the MYD88 L265P oncogenic mutation. IMO-8400, a clinical-stage drug candidate, was well tolerated in clinical trials conducted in healthy subjects and patients with psoriasis, having demonstrated no myelosuppression or any pattern of hematological or other toxicity in 12-week treatment regimens. The risk-benefit profile of IMO-8400 to date merits further clinical testing for genetically defined B-cell malignancies, as monotherapy and in combination with other agents. Based on the accumulated preclinical data, clinical safety, and demonstrated mechanism of action of IMO-8400, Phase 1/2 clinical trials of IMO-8400 are being conducted in both relapsed/refractory patients with WM and in patients with DLBCL and the MYD88 L265P mutation. DisclosuresBrenner:Idera Pharmaceuticals : Employment, Equity Ownership. Arbeit:Idera Pharmaceuticals: Employment. Sullivan:Idera Pharmaceuticals: Employment.

Genetic variations in MyD88 adaptor-like are associated with atopic dermatitis

Toll-like receptors (TLRs) are important pathogen-associated molecular pattern recognition receptors involved in initiating immune responses. The adaptor protein MyD88 adaptor-like (Mal), involved in signaling downstream of TLRs, plays a crucial role in mediating NF-κB activation. The association of Mal polymorphisms with allergic diseases has not previously been defined. The objective of this study was to detect polymorphisms in the Mal gene and to investigate their association with allergic diseases. Mal gene polymorphisms were genotyped in 310 subjects. The functional effects of Mal variants were analyzed in vitro. One Mal polymorphism, c.303 G>A (Q101Q), was found at a significantly lower frequency in atopic dermatitis patients (p=0.016). Q101Q is in linkage disequilibrium with -103 A>G (rs1893352) and c.539 C>T (S180L) (rs8177374) in the HapMap database. The A allele of -103 A>G showed significantly reduced transcription of Mal compared with the G allele. In addition, three rare variants were identified in this study, c.394 G>A (E132K), c.428 G>A (R143Q) and c.570 G>C (E190D), and were shown to lead to loss-of-function of Mal. It is possible that gene polymorphisms in Mal could affect atopic dermatitis by influencing the innate immune system. We show that Q101Q, which is in linkage disequilibrium with -103 A>G and S180L, may play a protective role against atopic dermatitis. Furthermore, we propose that loss-of-function variants of Mal could predispose individuals to atopic dermatitis or other immunological disorders.

Salmonella enterica strains belonging to O serogroup 1,3,19 induce chlorosis and wilting of Arabidopsis thaliana leaves

The number of outbreaks and illness linked to the consumption of contaminated salad leaves have increased dramatically in the last decade. Escherichia coli and Salmonella enterica are the most common food-borne pathogens linked to consumption of fresh produce. Different serovars of S. enterica subspecies enterica have been shown to bind the surface of salad leaves, to exhibit tropism towards the stomata and to invade leaves and reach the underlying mesophyll. However the consequences of leaf invasion are not known. Here we show that following infiltration, serovars Typhimurium, Enteritidis, Heidelberg and Agona, as well as strains of S. enterica subspecies arizonae and diarizonae, survive in the mesophyll of Arabidopsis thaliana leaves but induce neither leaf chlorosis nor wilting. In contrast, S. Senftenberg induced strong leaf wilting 4 days post infiltration in A. thaliana accession Col-0 but not in accession Ws-0. Dead S. Senftenberg and bacterial lysates also induced leaf wilting. We found that mutations in the Arabidopsis pathogen associated molecular pattern (PAMP) recognition receptors (PRRs) FLS2, which recognizes flagellin, and EFR, which recognizes the bacterial elongation factor EF-Tu, had no effect on the wilting response of A. thaliana to S. Senftenberg. Infiltration of A. thaliana leaves with serovars Cannstatt, Krefeld and Liverpool, which like Senftenberg belong to Salmonella serogroup E(4) (O:1,3,19), also resulted in rapid leaf wilting, while all tested rough S. Senftenberg strains (lacking the O antigen) failed to elicit leaf wilting. These results suggest that the Salmonella O antigen 1,3,19 specifically triggers leaf chlorosis and wilting in A. thaliana.

The coming of age of virus-like particle vaccines

Virus-like particles are supra-molecular assemblages, usually icosahedral or rod-like structures. They incorporate key immunologic features of viruses which include repetitive surfaces, particulate structures and induction of innate immunity through activation of pathogen-associated molecular-pattern recognition receptors. They carry no replicative genetic information and can be produced recombinantly in large scale. Virus-like particles thus represent a safe and effective vaccine platform for inducing potent B- and T-cell responses. In addition to being effective vaccines against the corresponding virus from which they are derived, virus-like particles can also be used to present foreign epitopes to the immune system. This can be achieved by genetic fusion or chemical conjugation. This technological innovation has greatly broadened the scope of their use, from immunizing against microbial pathogens to immunotherapy for chronic diseases. Towards this end, virus-like particles have been used to induce autoantibodies to disease-associated self-molecules involved in chronic diseases, such as hypertension and Alzheimer's disease. The recognition of the potent immunogenicity and commercial potential for virus-like particles has greatly accelerated research and development activities. During the last decade, two prophylactic virus-like particle vaccines have been registered for human use, while another 12 vaccines entered clinical development.

Characterization, expression and evolution analysis of Toll‐like receptor 1 gene in pufferfish (Tetraodon nigroviridis)

The Toll-like receptor (TLR) family is a pathogen-associated molecular pattern recognition receptor family that plays key roles in the activation of innate immune system. As an important member of TLRs, TLR1 is involved in the recognition of triacylated lipoproteins and mycobacterial products by binding to TLR2 to form a heterodimer. Although partial sequences of TLR1 has been predicted in Fugu and Danio by bioinformatics, the molecular cloning and characterization of this gene in fish still remains largely unclear. Here we report the full-length sequence of TLR1 gene in the pufferfish (Tetraodon nigroviridis), providing experimental evidence to its existence in fish. The cloned Tetraodon TLR1 (TnTLR1) cDNA exhibited 2587 bp in length and contains a 122-bp 5' untranslated region (UTR), a 2391-bp open reading frame and a 74-bp 3' UTR. Expression of TLR1 transcripts in most selected tissues were detected at lower levels; however, the transcripts in spleen was markedly increased (threefold) after stimulation with lipopolysaccharide, indicating that TLR1 in fish may be involved in the inflammatory responses to bacterial endotoxin or Gram-negative bacteria infection. Gene organization of TnTLR1 is similar to mammalian and avian, and a high degree of conservation of chromosome synteny exists between them. The protein sequence shares 33.7-77.3% identity with known TLR1 members. Phylogenetic analysis grouped TnTLR1 with other fish TLRs on a separated branch, excluded from mammalian and avian TLR1s. TnTLR1 with 796 amino acids has a calculated molecular mass of 90.69 kDa and a theoretical pI of 8.43. Structurally, TnTLR1 has a Toll/interleukin-1 receptor (TIR) domain, a transmembrane domain and leucine-rich repeat domain which are the hallmarks of TLR family. Phylogenetic analysis provided evidence that the TnTLR1 was very close to mammalian TLR1, TLR6 and TLR10 and seemed to be their common ancestor. This report of the full-length TLR1 gene in Tetraodon will provide us further insights into the study of both function and evolution of fish TLRs as a whole.

Immune activation suppresses initiation of lytic Epstein-Barr virus infection

Primary infection with Epstein-Barr virus (EBV) is asymptomatic in children with immature immune systems but may manifest as infectious mononucleosis, a vigorous immune activation, in adolescents or adults with mature immune systems. Infectious mononucleosis and chronic immune activation are linked to increased risk for EBV-associated lymphoma. Here we show that EBV initiates progressive lytic infection by expression of BZLF-1 and the late lytic genes gp85 and gp350/220 in cord blood mononuclear cells (CBMC) but not in peripheral blood mononuclear cells (PBMC) from EBV-naive adults after EBV infection ex vivo. Lower levels of proinflammatory cytokines in CBMC, used to model a state of minimal immune activation and immature immunity, than in PBMC were associated with lytic EBV infection. Triggering the innate immunity specifically via Toll-like receptor-9 of B cells substantially suppressed BZLF-1 mRNA expression in acute EBV infection ex vivo and in anti-IgG-stimulated chronically latently EBV-infected Akata Burkitt lymphoma cells. This was mediated in part by IL-12 and IFN-gamma. These results identify immune activation as critical factor for the suppression of initiation of lytic EBV infection. We hypothesize that immune activation contributes to EBV-associated lymphomagenesis by suppressing lytic EBV and in turn promotes latent EBV with transformation potential.

N-(3-Oxo-acyl)homoserine Lactones Signal Cell Activation through a Mechanism distinct from the Canonical Pathogen-associated Molecular Pattern Recognition Receptor Pathways

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). Here we have shown that p38 and eIF2alpha phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa.

Modulating responsiveness of human TLR7 and 8 to small molecule ligands with T‐rich phosphorothiate oligodeoxynucleotides

Toll-like receptors (TLR) 7 and 8 are closely related members of the TLR family of pathogen-associated molecular pattern recognition receptors and have an important function in activation of innate immune responses upon viral infection. TLR7 can be activated selectively by the guanosine analogue loxoribine, whereas the imidazoquinoline derivative Resiquimod (R-848) activates both TLR7 and TLR8. We demonstrate that co-incubation of R-848 with thymidine homopolymer oligodeoxynucleotides (ODN) significantly increased activity of R-848 on TLR8-expressing HEK 293 cells, but abolished TLR7-mediated signaling. Similarly, the combination of loxoribine and thymidine ODN redirected the stimulatory effect of loxoribine away from TLR7, and toward TLR8. This alteration in ligand specificity was demonstrated both in TLR-transfected HEK cells, and also in human PBMC, with a corresponding change in cytokine production away from IFN-alpha secretion by TLR7-expressing plasmacytoid DC and toward IL-12, TNF-alpha and IFN-gamma secretion by TLR8-expressing monocytes and NK cells. These results demonstrate an unexpected plasticity in the ligand specificities of TLR7 and TLR8, and suggest a novel sequence-selective interaction between these receptors and synthetic phosphorothioate ODN.

Hyperconservation of the N-formyl peptide binding site of M3: evidence that M3 is an old eutherian molecule with conserved recognition of a pathogen-associated molecular pattern.

The mouse MHC class I-b molecule H2-M3 has unique specificity for N-formyl peptides, derived from bacteria (and mitochondria), and is thus a pathogen-associated molecular pattern recognition receptor (PRR). To test whether M3 was selected for this PRR function, we studied M3 sequences from diverse murid species of murine genera Mus, Rattus, Apodemus, Diplothrix, Hybomys, Mastomys, and Tokudaia and of sigmodontine genera Sigmodon and PEROMYSCUS: We found that M3 is highly conserved, and the 10 residues coordinating the N-formyl group are almost invariant. The ratio of nonsynonymous and synonymous substitution rates suggests the Ag recognition site of M3, unlike the Ag recognition site of class I-a molecules, is under strong negative (purifying) selection and has been for at least 50-65 million years. Consistent with this, M3 alpha1alpha2 domains from Rattus norvegicus and Sigmodon hispidus and from the "null" allele H2-M3(b) specifically bound N-formyl peptides. The pattern of nucleotide substitution in M3 suggests M3 arose rapidly from murid I-a precursors by an evolutionary leap ("saltation"), perhaps involving intense selective pressure from bacterial pathogens. Alternatively, M3 arose more slowly but prior to the radiation of eutherian (placental) mammals. Older dates for the emergence of M3, and the accepted antiquity of CD1, suggest that primordial class I MHC molecules could have evolved originally as monomorphic PRR, presenting pathogen-associated molecular patterns. Such MHC PRR molecules could have been preadaptations for the evolution of acquired immunity during the early vertebrate radiation.

Gm-CSF regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense.

Recent studies in transgenic mice have revealed important insights into the roles of GM-CSF in regulation of surfactant homeostasis and lung host defense. Interruption of the GM-CSF signaling pathway by targeted ablation of the GM-CSF gene or its receptor (GM(-/-) or GM Rbetac(-/-) mice, respectively) resulted in pulmonary alveolar proteinosis (PAP) but no hematologic abnormalities. Alveolar macrophages from GM(-/-) mice have reduced capacity for surfactant catabolism, cell adhesion, phagocytosis, bacterial killing, Toll-receptor signaling, and expression of various pathogen-associated molecular pattern recognition receptors, suggesting arrest at an early stage of differentiation. PAP and abnormalities of alveolar macrophage function were corrected by local expression of GM-CSF in the lung, and expression of the transcription factor PU.1 in alveolar macrophages of GM(-/-) mice rescued most defects. Recently, a strong association of auto-antibodies to GM-CSF or GM-CSF receptor gene mutations with PAP has implicated GM-CSF signaling abnormalities in the pathogenesis of PAP in humans. Together, these observations demonstrate that GM-CSF has a critical role in regulation of surfactant homeostasis and alveolar macrophage innate immune functions in the lung.