Loss of SP-A in the Lung Exacerbates Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating and common chronic lung disease that is pathologically characterized by the destruction of lung architecture and the accumulation of extracellular matrix in the lung. Previous studies have shown an association between lung surfactant protein (SP) and the pathogenesis of IPF, as demonstrated by mutations and the altered expression of SP in patients with IPF. However, the role of SP in the development of lung fibrosis is poorly understood. In this study, the role of surfactant protein A (SP-A) was explored in experimental lung fibrosis induced with a low or high dose of bleomycin (BLM) and CRISPR/Cas9-mediated genetic deletion of SP-A. Our results showed that lung SP-A deficiency in mice promoted the development of fibrotic damage and exacerbated inflammatory responses to the BLM challenge. In vitro experiments with murine lung epithelial LA-4 cells demonstrated that in response to transforming growth factor-ß1 (TGF-ß1), LA-4 cells had a decreased protein expression of SP-A. Furthermore, exogenous SP administration to LA-4 cells inhibited the TGF-ß1-induced upregulation of fibrotic markers. Overall, these findings suggest a novel antifibrotic mechanism of SP-A in the development of lung fibrosis, which indicates the therapeutic potential of the lung SP-A in preventing the development of IPF.

New Directions in Pulmonary Gene Therapy.

The lung has long been a target for gene therapy, yet efficient delivery and phenotypic disease correction has remained challenging. Although there have been significant advancements in gene therapies of other organs, including the development of several ex vivo therapies, in vivo therapeutics of the lung have been slower to transition to the clinic. Within the past few years, the field has witnessed an explosion in the development of new gene addition and gene editing strategies for the treatment of monogenic disorders. In this review, we will summarize current developments in gene therapy for cystic fibrosis, alpha-1 antitrypsin deficiency, and surfactant protein deficiencies. We will explore the different gene addition and gene editing strategies under investigation and review the challenges of delivery to the lung.

A 20-Mer Peptide Derived from the Lectin Domain of SP-A2 Decreases Tumor Necrosis Factor Alpha Production during Mycoplasma pneumoniae Infection.

Human surfactant protein-A2 (hSP-A2) is a component of pulmonary surfactant that plays an important role in the lung's immune system by interacting with viruses, bacteria, and fungi to facilitate pathogen clearance and by downregulating inflammatory responses after an allergic challenge. Genetic variation in SP-A2 at position Gln223Lys is present in up to ∼30% of the population and has been associated with several lung diseases, such as asthma, pulmonary fibrosis, and lung cancer (M. M. Pettigrew, J. F. Gent, Y. Zhu, E. W. Triche, et al., BMC Med Genet 8:15, 2007, https://bmcmedgenet.biomedcentral.com/articles/10.1186/1471-2350-8-15; Y. Wang, P. J. Kuan, C. Zing, J. T. Cronkhite, et al., Am J Hum Genet 84:52-59, 2009, https://www.cell.com/ajhg/fulltext/S0002-9297(08)00595-8). Previous work performed by our group showed differences in levels of SP-A binding to non-live mycoplasma membrane fractions that were dependent on the presence of a lysine (K) or a glutamine (Q) at amino acid position 223 in the carbohydrate region of SP-A2. On the basis of these differences, we have derived 20-amino-acid peptides flanking this region of interest in order to test the ability of each to regulate various immune responses to live Mycoplasma pneumoniae in SP-A knockout mice and RAW 264.7 cells. In both models, the 20-mer containing 223Q significantly decreased both tumor necrosis factor alpha (TNF-α) mRNA levels and protein levels in comparison to the 20-mer containing 223K during M. pneumoniae infection. While neither of the 20-mer peptides (223Q and 223K) had an effect on p38 phosphorylation during M. pneumoniae infection, the 223Q-20mer peptide significantly reduced NF-κB p65 phosphorylation in both models. Taken together, our data suggest that small peptides derived from the lectin domain of SP-A2 that contain the major allelic variant (223Q) maintain activity in reducing TNF-α induction during M. pneumoniae infection.

Surfactant protein A deficiency exacerbates renal interstitial fibrosis following obstructive injury in mice.

Renal interstitial fibrosis is an inevitable consequence of virtually every type of chronic kidney disease. The underlying mechanisms, however, are not completely understood. In the present study, we identified surfactant protein A (SP-A) as a novel protein factor involved in the renal fibrosis induced by unilateral ureter obstruction (UUO). UUO induced SP-A expression in mouse kidney epithelium, likely due to the increased acidic stress and inflammation. Interestingly, SP-A deficiency aggravated UUO-prompted kidney structural damage, macrophage accumulation, and tubulointerstitial fibrosis. SP-A deficiency appeared to worsen the fibrosis by enhancing interstitial myofibroblast accumulation. Moreover, SP-A deficiency increased the expression of TGF-ß1, the major regulator of kidney fibrosis, particularly in the interstitial cells. Mechanistically, SP-A deficiency increased the expression and release of high mobility group box 1 (HMGB1), a factor regulating TGF-ß expression/signaling and implicated in renal fibrosis. SP-A also blocked HMGB1 activities in inducing TGF-ß1 expression and myofibroblast transdifferentiation from kidney fibroblasts, demonstrating that SP-A protected kidney by impeding both the expression and fibrogenic function of HMGB1. Since SP-A physically interacted with HMGB1 both in vitro and in kidney tissue in vivo, SP-A may exert its protective role by binding to HMGB1 and thus titrating its activity during UUO-induced renal fibrosis.

Steroid receptor coactivators 1 and 2 mediate fetal-to-maternal signaling that initiates parturition.

The precise mechanisms that lead to parturition are incompletely defined. Surfactant protein-A (SP-A), which is secreted by fetal lungs into amniotic fluid (AF) near term, likely provides a signal for parturition; however, SP-A-deficient mice have only a relatively modest delay (~12 hours) in parturition, suggesting additional factors. Here, we evaluated the contribution of steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2), which upregulate SP-A transcription, to the parturition process. As mice lacking both SRC-1 and SRC-2 die at birth due to respiratory distress, we crossed double-heterozygous males and females. Parturition was severely delayed (~38 hours) in heterozygous dams harboring SRC-1/-2-deficient embryos. These mothers exhibited decreased myometrial NF-κB activation, PGF2α, and expression of contraction-associated genes; impaired luteolysis; and elevated circulating progesterone. These manifestations also occurred in WT females bearing SRC-1/-2 double-deficient embryos, indicating that a fetal-specific defect delayed labor. SP-A, as well as the enzyme lysophosphatidylcholine acyltransferase-1 (LPCAT1), required for synthesis of surfactant dipalmitoylphosphatidylcholine, and the proinflammatory glycerophospholipid platelet-activating factor (PAF) were markedly reduced in SRC-1/-2-deficient fetal lungs near term. Injection of PAF or SP-A into AF at 17.5 days post coitum enhanced uterine NF-κB activation and contractile gene expression, promoted luteolysis, and rescued delayed parturition in SRC-1/-2-deficient embryo-bearing dams. These findings reveal that fetal lungs produce signals to initiate labor when mature and that SRC-1/-2-dependent production of SP-A and PAF is crucial for this process.

Surfactant-associated proteins: structure, function and clinical implications.

Surfactant replacement therapy is now the standard of care for infants with respiratory distress syndrome. As the understanding of surfactant structure and function has evolved, surfactant-associated proteins are now understood to be essential components of pulmonary surfactant. Their structural and functional diversity detail the complexity of their contributions to normal pulmonary physiology, and deficiency states result in significant pathology. Engineering synthetic surfactant protein constructs has been a major research focus for replacement therapies. This review highlights what is known about surfactant proteins and how this knowledge is pivotal for future advancements in treating respiratory distress syndrome as well as other pulmonary diseases characterized by surfactant deficiency or inactivation.

Enhanced liver autophagic activity improves survival of septic mice lacking surfactant proteins A and D.

Autophagy is a protective cellular mechanism in response to various stresses, including sepsis. Sepsis is defined as systemic inflammation by infection. Surfactant protein A and D (SP-A and SP-D) are involved in host defense, regulation of inflammation, and homeostasis, but their roles in the autophagic activity and relevant gene expression in sepsis are unclear. In this study, mice lacking SP-A and SP-D (SP-A/D KO mice) and background-matched wild-type (WT) C57BL/6 mice underwent either cecal ligation and puncture (CLP) or sham surgery. The results showed that SP-A/D KO mice had lower mortality than WT mice in CLP sepsis. Liver tissues showed marked pathological changes in both septic SP-A/D KO and WT mice 24 hrs after CLP treatment; and quantitative analysis of liver histopathology revealed significant difference between septic SP-A/D and septic WT mice. SP-A/D KO mice had higher basal and sepsis-induced level of autophagy than WT mice (p < 0.05), as judged by Western blot and electron microscopic analyses. The expression of 84 autophagy-related genes revealed differential basal and sepsis-induced gene expression between SP-A/D KO and WT mice. The expression increased in three genes and decreased in four genes in septic WT mice, as compared to septic SP-A/D KO mice (p < 0.05). Furthermore, differential responses to sepsis between SP-A/D KO and WT mice were found in six signaling pathways related to autophagy and apoptosis. Therefore, enhanced autophagic activity improves the survival of septic SP-A/D KO mice through the regulation of liver autophagy/apoptosis-related gene expression and signaling pathway activation.

Deficiency in pulmonary surfactant proteins in mice with fatty acid binding protein 4-Cre-mediated knockout of the tuberous sclerosis complex 1 gene.

Tuberous sclerosis complex 1 (TSC1) forms a heterodimmer with tuberous sclerosis complex 2, to inhibit signalling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). The mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as gene transcription and protein translation, in response to growth factors and nutrient signals. Originally designed to test the role of TSC1 in adipocyte function, mice in which the gene for TSC1 was specifically deleted by the fatty acid binding protein 4 (FABP4)-Cre (Fabp4-Tsc1cKO mice) died prematurely within 48 h after birth. The Fabp4-Tsc1cKO mouse revealed a much smaller phenotype relative to the wild-type littermates. Maternal administration of rapamycin, a classical mTOR inhibitor, significantly increased the survival time of Fabp4-Tsc1cKO mice for up to 23 days. Both macroscopic and microscopic haemorrhages were observed in the lungs of Fabp4-Tsc1cKO mice, while other tissues showed no significant changes. Levels of surfactant proteins A and B demonstrated a significant decrease in the Fabp4-Tsc1cKO mice, which was rescued by maternal injection of rapamycin. Co-localization of FABP4 or TSC1 with surfactant protein B was also detected in neonatal pulmonary tissues. Our study suggests that TSC1-mTORC1 may be critical for the synthesis of surfactant proteins A and B.

Mast cell TNF receptors regulate responses to Mycoplasma pneumoniae in surfactant protein A (SP-A)-/- mice.

BACKGROUND: Mycoplasma pneumoniae (Mp) frequently colonizes the airways of patients with chronic asthma and likely contributes to asthma exacerbations. We previously reported that mice lacking surfactant protein A (SP-A) have increased airway hyperresponsiveness (AHR) during M pneumoniae infection versus wild-type mice mediated by TNF-α. Mast cells (MCs) have been implicated in AHR in asthma models and produce and respond to TNF-α. OBJECTIVE: Determine the contribution of MC/TNF interactions to AHR in airways lacking functional SP-A during Mp infection. METHODS: Bronchoalveolar lavage fluid was collected from healthy and asthmatic subjects to examine TNF-α levels and M pneumoniae positivity. To determine how SP-A interactions with MCs regulate airway homeostasis, we generated mice lacking both SP-A and MCs (SP-A(-/-)Kit(W-sh/W-sh)) and infected them with M pneumoniae. RESULTS: Our findings indicate that high TNF-α levels correlate with M pneumoniae positivity in human asthmatic patients and that human SP-A inhibits M pneumoniae-stimulated transcription and release of TNF-α by MCs, implicating a protective role for SP-A. MC numbers increase in M pneumoniae-infected lungs, and airway reactivity is dramatically attenuated when MCs are absent. Using SP-A(-/-)Kit(W-sh/W-sh) mice engrafted with TNF-α(-/-) or TNF receptor (TNF-R)(-/-) MCs, we found that TNF-α activation of MCs through the TNF-R, but not MC-derived TNF-α, leads to augmented AHR during M pneumoniae infection when SP-A is absent. Additionally, M pneumoniae-infected SP-A(-/-)Kit(W-sh/W-sh) mice engrafted with TNF-α(-/-) or TNF-R(-/-) MCs have decreased mucus production compared with that seen in mice engrafted with wild-type MCs, whereas burden was unaffected. CONCLUSION: Our data highlight a previously unappreciated but vital role for MCs as secondary responders to TNF-α during the host response to pathogen infection.

Novel role for surfactant protein A in gastrointestinal graft-versus-host disease.

Graft-versus-host disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal (GI) tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host Ags as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT. C57BL/6 (H2b; WT) and SP-A-deficient mice on a C57BL/6 background (H2b; SP-A(-/-)) mice underwent allogeneic or syngeneic BMT with cells from either C3HeB/FeJ (H2k; SP-A-deficient recipient mice that have undergone an allogeneic BMT [SP-A(-/-)alloBMT] or SP-A-sufficient recipient mice that have undergone an allogeneic BMT) or C57BL/6 (H2b; SP-A-deficient recipient mice that have undergone a syngeneic BMT or SP-A-sufficient recipient mice that have undergone a syngeneic BMT) mice. Five weeks post-BMT, mice were necropsied, and lung and GI tissue were analyzed. SP-A(-/-) alloBMT or SP-A-sufficient recipient mice that have undergone an allogeneic BMT had no significant differences in lung pathology; however, SP-A(-/-)alloBMT mice developed marked features of GI GVHD, including decreased body weight, increased tissue inflammation, and lymphocytic infiltration. SP-A(-/-)alloBMT mice also had increased colon expression of IL-1ß, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells and diminished regulatory T cells. Our results demonstrate the first evidence, to our knowledge, of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an allogeneic BMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased regulatory T cells. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.

Impact of sex and ozone exposure on the course of pneumonia in wild type and SP-A (-/-) mice.

Female mice exhibited higher survival rate than males after pneumonia, with a reversal of this pattern following ozone exposure. Surfactant protein A (SP-A) plays an important role in innate immunity and SP-A (-/-) mice were more susceptible to pneumonia than wild type mice. Here, we investigated underlying mechanisms of the differential susceptibility of mice to pneumonia. Wild type and SP-A (-/-) C57BL/6J male and female mice were exposed to ozone or filtered air (FA) and then infected intratracheally with Klebsiella pneumoniae. Blood, spleen, and lung were analyzed for bacterial counts, lung and spleen weights, and sex hormone and cortisol levels were measured in plasma within two days post-infection. We found: 1) in the absence of ozone-induced oxidative stress, males had higher level of bacterial dissemination compared to females; ozone exposure decreased pulmonary clearance in both sexes and ozone-exposed females were more affected than males; 2) ozone exposure increased lung weight, but decreased spleen weight in both sexes, and in both cases ozone-exposed females were affected the most; 3) plasma cortisol levels in infected mice changed: ozone-exposed>FA-exposed, females>males, and infected>non-infected; 4) no major sex hormone differences were observed in the studied conditions; 5) differences between wild type and SP-A (-/-) mice were observed in some of the studied conditions. We concluded that reduced pulmonary clearance, compromised spleen response to infection, and increased cortisol levels in ozone-exposed females, and the higher level of lung bacterial dissemination in FA-exposed males, contribute to the previously observed survival outcomes.

Surfactant protein A integrates activation signal strength to differentially modulate T cell proliferation.

Pulmonary surfactant lipoproteins lower the surface tension at the alveolar-airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A-mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A(-/-) mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A-dependent effects are mediated by changes in intracellular Ca(2+) levels over time, involving extrinsic Ca(2+)-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation.

Pulmonary surfactant protein A enhances endolysosomal trafficking in alveolar macrophages through regulation of Rab7.

Surfactant protein A (SP-A), the most abundant pulmonary soluble collectin, modulates innate and adaptive immunity of the lung, partially via its direct effects on alveolar macrophages (AM), the most predominant intra-alveolar cells under physiological conditions. Enhanced phagocytosis and endocytosis are key functional consequences of AM/SP-A interaction, suggesting a SP-A-mediated modulation of small Rab (Ras related in brain) GTPases that are pivotal membrane organizers in both processes. In this article, we show that SP-A specifically and transiently enhances the protein expression of endogenous Rab7 and Rab7b, but not Rab5 and Rab11, in primary AM from rats and mice. SP-A-enhanced GTPases are functionally active as determined by increased interaction of Rab7 with its downstream effector Rab7 interacting lysosomal protein (RILP) and enhanced maturation of cathepsin-D, a function of Rab7b. In AM and RAW264.7 macrophages, the SP-A-enhanced lysosomal delivery of GFP-Escherichia coli is abolished by the inhibition of Rab7 and Rab7 small interfering RNA transfection, respectively. The constitutive expression of Rab7 in AM from SP-A(-/-) mice is significantly reduced compared with SP-A(+/+) mice and is restored by SP-A. Rab7 blocking peptides antagonize SP-A-rescued lysosomal delivery of GFP-E. coli in AM from SP-A(-/-) mice. Activation of Rab7, but not Rab7b, by SP-A depends on the PI3K/Akt/protein kinase Cζ (PKCζ) signal transduction pathway in AM and RAW264.7 macrophages. SP-A induces a Rab7/PKCζ interaction in these cells, and the disruption of PKCζ by small interfering RNA knockdown abolishes the effect of SP-A on Rab7. The data demonstrate a novel role for SP-A in modulating endolysosomal trafficking via Rab7 in primary AM and define biochemical pathways involved.

Dispensability of surfactant proteins A and D in immune control of Mycobacterium tuberculosis infection following aerosol challenge of mice.

Surfactant proteins A and D (SP-A and -D) play a role in many acute bacterial, viral, and fungal infections and in acute allergic responses. In vitro, human SPs bind Mycobacterium tuberculosis and alter human and rat macrophage-mediated functions. Here we report the roles of SP-A and SP-D in M. tuberculosis infection following aerosol challenge of SP-A-, SP-D-, and SP-A/-D-deficient mice. These studies surprisingly identified no gross defects in uptake or immune control of M. tuberculosis in SP-A-, SP-D-, and SP-A/-D-deficient mice. While both SP-A- and SP-D-deficient mice exhibited evidence of immunopathologic defects, the CD11b(high) CD11c(high) dendritic cell populations and the gamma interferon (IFN-γ)-dependent CD4(+) T cell response to M. tuberculosis were unaltered in all genotypes tested. Together, these data indicate that SP-A and SP-D are dispensable for immune control of M. tuberculosis in a low-dose, aerosol challenge, murine model of tuberculosis (TB).

Lung effector memory and activated CD4+ T cells display enhanced proliferation in surfactant protein A-deficient mice during allergen-mediated inflammation.

Although many studies have shown that pulmonary surfactant protein (SP)-A functions in innate immunity, fewer studies have addressed its role in adaptive immunity and allergic hypersensitivity. We hypothesized that SP-A modulates the phenotype and prevalence of dendritic cells (DCs) and CD4(+) T cells to inhibit Th2-associated inflammatory indices associated with allergen-induced inflammation. In an OVA model of allergic hypersensitivity, SP-A(-/-) mice had greater eosinophilia, Th2-associated cytokine levels, and IgE levels compared with wild-type counterparts. Although both OVA-exposed groups had similar proportions of CD86(+) DCs and Foxp3(+) T regulatory cells, the SP-A(-/-) mice had elevated proportions of CD4(+) activated and effector memory T cells in their lungs compared with wild-type mice. Ex vivo recall stimulation of CD4(+) T cell pools demonstrated that cells from the SP-A(-/-) OVA mice had the greatest proliferative and IL-4-producing capacity, and this capability was attenuated with exogenous SP-A treatment. Additionally, tracking proliferation in vivo demonstrated that CD4(+) activated and effector memory T cells expanded to the greatest extent in the lungs of SP-A(-/-) OVA mice. Taken together, our data suggested that SP-A influences the prevalence, types, and functions of CD4(+) T cells in the lungs during allergic inflammation and that SP deficiency modifies the severity of inflammation in allergic hypersensitivity conditions like asthma.

Nitric oxide mediates relative airway hyporesponsiveness to lipopolysaccharide in surfactant protein A-deficient mice.

Surfactant protein A (SP-A) mediates innate immune cell responses to LPS, a cell wall component of gram-negative bacteria that is found ubiquitously in the environment and is associated with adverse health effects. Inhaled LPS induces lung inflammation and increases airway responsiveness (AR). However, the role of SP-A in mediating LPS-induced AR is not well-defined. Nitric oxide (NO) is described as a potent bronchodilator, and previous studies showed that SP-A modulates the LPS-induced production of NO. Hence, we tested the hypothesis that increased AR, observed in response to aerosolized LPS exposure, would be significantly reduced in an SP-A-deficient condition. Wild-type (WT) and SP-A null (SP-A(-/-)) mice were challenged with aerosolized LPS. Results indicate that despite similar inflammatory indices, LPS-treated SP-A(-/-) mice had attenuated AR after methacholine challenge, compared with WT mice. The attenuated AR could not be attributed to inherent differences in SP-D concentrations or airway smooth muscle contractile and relaxation properties, because these measures were similar between WT and SP-A(-/-) mice. LPS-treated SP-A(-/-) mice, however, had elevated nitrite concentrations, inducible nitric oxide synthase (iNOS) expression, and NOS activity in their lungs. Moreover, the administration of the iNOS-specific inhibitor 1400W completely abrogated the attenuated AR. Thus, when exposed to aerosolized LPS, SP-A(-/-) mice demonstrate a relative airway hyporesponsiveness that appears to be mediated at least partly via an iNOS-dependent mechanism. These findings may have clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a variety of lung diseases.

Susceptibility of mice genetically deficient in SP-A or SP-D gene to invasive pulmonary aspergillosis.

Pulmonary surfactant proteins, SP-A and SP-D, are carbohydrate pattern recognition molecules of innate immunity, which significantly enhance phagocytosis and killing of Aspergillus fumigatus, a pathogenic fungus, by neutrophils and macrophages. The present study examined the susceptibility of immunosuppressed SP-A gene deficient (SP-A(-/-)) or SP-D gene deficient (SP-D(-/-)) mice to A. fumigatus conidia challenge compared to wild-type (WT) mice. A. fumigatus-challenged SP-A(-/-) (SP-A(-/-) IPA) mice showed less mortality (40%) than the WT-IPA mice (100%) and increased mortality (60%) following administration of SP-A with decreased TNF-alpha and IFN-gamma to IL-4 ratio than SP-A(-/-) IPA mice. The SP-D(-/-) IPA mice (57.14%) showed similar mortality as WT-IPA mice (60%). However, the SP-D (-/-) IPA mice (42.86% mortality on day 2) died earlier than the WT-IPA mice (20% mortality on day 2), showed a higher hyphal density and tissue injury in lungs. Treatment with SP-D or a recombinant fragment of human SP-D rhSP-D reduced the mortality to 50% and 33%, respectively, concomitant with higher IFN-gamma to IL-4 ratios in treated SP-D(-/-) mice, compared to untreated control group. The results showed that SP-D gene deficient mice are more susceptible to IPA while SP-A gene deficient mice acquire resistance to IPA.

Surfactant protein (SP)-A and SP-D as antimicrobial and immunotherapeutic agents.

Surfactant protein (SP)-A and SP-D belong to the "Soluble C-type Lectin" family of proteins and are collectively known as "Collectins". Based on their ability to recognize pathogens and to regulate the host defense, SP-A and SP-D have been recently categorized as "Secretory Pathogen Recognition Receptors". SP-A and SP-D were first identified in the lung; the expression of SP-A and SP-D has also been observed at other mucosal surfaces, such as lacrimal glands, gastrointestinal mucosa, genitourinary epithelium and periodontal surfaces. Since the role of these proteins is not fully elucidated at other mucosal surfaces, the focus of this article is on lung-SP-A and SP-D. It has become clear from research studies performed over a number of years that SP-A and SP-D are critical for the maintenance of lung homeostasis and the regulation of host defense and inflammation. However, none of the surfactant preparations available for clinical use have SP-A or SP-D. A review is presented here on SP-A- and SP-D-deficiencies in lung diseases, the importance of the administration of SP-A and SP-D, and recent patents and research directions that may lead to the design of novel SP-A- or SP-D-based therapeutics and surfactants.

Pulmonary pathology in thyroid transcription factor-1 deficiency syndrome.

Thyroid transcription factor-1 (TTF-1) deficiency syndrome is characterized by neurologic, thyroidal, and pulmonary dysfunction. Children usually have mild-to-severe respiratory symptoms and occasionally die of respiratory failure. Herein, we describe an infant with a constitutional 14q12-21.3 haploid deletion encompassing the TTF-1 gene locus who had cerebral dysgenesis, thyroidal dysfunction, and respiratory insufficiency. The clinical course was notable for mild hyaline membrane disease, continuous ventilatory support, and symmetrically distributed pulmonary cysts by imaging. He developed pneumonia and respiratory failure and died at 8 months. Pathologically, the lungs had grossly visible emphysematous changes with "cysts" up to 2 mm in diameter. The airway generations and radial alveolar count were diminished. In addition to acute bacterial pneumonia, there was focally alveolar septal fibrosis, pneumocyte hypertrophy, and clusters of airspace macrophages. Ultrastructurally, type II pneumocytes had numerous lamellar bodies, and alveolar spaces contained fragments of type II pneumocytes and extruded lamellar bodies. Although immunoreactivity for surfactant protein SP-A and ABCA3 was diminished, that for SP-B and proSP-C was robust, although irregularly distributed, corresponding to the distribution of type II pneumocytes. Immunoreactivity for TTF-1 protein was readily detected. In summation, we document abnormal airway and alveolar morphogenesis and altered expression of surfactant-associated proteins, which may explain the respiratory difficulties encountered in TTF-1 haploinsufficiency. These findings are consistent with experimental evidence documenting the important role of TTF-1 in pulmonary morphogenesis and surfactant metabolism.

Humanized SFTPA1 and SFTPA2 transgenic mice reveal functional divergence of SP-A1 and SP-A2: formation of tubular myelin in vivo requires both gene products.

Surfactant protein A (SP-A) plays a role in lung innate immunity and surfactant-related functions. Two functional genes, SP-A1 (SFTPA1) and SP-A2 (SFTPA2), are present in humans and primates (rodents have one gene). Single gene SP-A1 or SP-A2 proteins expressed in vitro are functional. To study their role in vivo, we generated humanized transgenic (hTG) C57BL/6 mice, SP-A1(6A(4)) and SP-A2(1A(3)). The SP-A cDNA in experimental constructs was driven by the 3.7-kb SP-C promoter. Positive hTG mice were bred with SP-A knock-out mice to generate F8 offspring for study. Epithelial alveolar type II cells were SP-A-positive, and Clara cells were negative by immunohistochemistry in hTG mice. The levels of SP-A in lungs of two hTG lines used were comparable with those in human lungs. Southern blot analysis indicated that two cDNA copies of either SP-A1(6A(4)) or SP-A2(1A(3)) were integrated as a concatemer into the genome of each of the two hTG lines. Electron microscopy analysis revealed that hTG mice with a single SP-A1(6A(4)) or SP-A2(1A(3)) gene product lacked tubular myelin (TM), but hTG mice carrying both had TM. Furthermore, TM was observed in human bronchoalveolar lavage fluid only if both SP-A1 and SP-A2 gene products were present and not in those containing primarily (>99.7%) either SP-A1 or SP-A2 gene products. In vivo rescue study confirmed that TM can only be restored after administering exogenous SP-A containing both SP-A1 and SP-A2 into the lungs of SP-A knock-out mice. These observations indicate that SP-A1 and SP-A2 diverged functionally at least in terms of TM formation.