Lung Remodeling Regions in Long-Term Coronavirus Disease 2019 Feature Basal Epithelial Cell Reprogramming.

Respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can trigger chronic lung disease that persists and even progresses after expected clearance of infectious virus. To gain an understanding of this process, the current study examined a series of consecutive fatal cases of coronavirus disease 2019 (COVID-19) that came to autopsy at 27 to 51 days after hospital admission. In each patient, a stereotyped bronchiolar-alveolar pattern of lung remodeling was identified with basal epithelial cell hyperplasia, immune activation, and mucinous differentiation. Remodeling regions featured macrophage infiltration and apoptosis and a marked depletion of alveolar type 1 and 2 epithelial cells. This pattern closely resembled findings from an experimental model of post-viral lung disease that requires basal-epithelial stem cell growth, immune activation, and differentiation. Together, these results provide evidence of basal epithelial cell reprogramming in long-term COVID-19 and thereby yield a pathway for explaining and correcting lung dysfunction in this type of disease.

Lung remodeling regions in long-term Covid-19 feature basal epithelial cell reprogramming.

Respiratory viruses, including SARS-CoV-2, can trigger chronic lung disease that persists and even progresses after expected clearance of infectious virus. To gain an understanding of this process, we examined a series of consecutive fatal cases of Covid-19 that came to autopsy at 27-51 d after hospital admission. In each patient, we identify a stereotyped bronchiolar-alveolar pattern of lung remodeling with basal epithelial cell hyperplasia and mucinous differentiation. Remodeling regions also feature macrophage infiltration and apoptosis and a marked depletion of alveolar type 1 and 2 epithelial cells. This entire pattern closely resembles findings from an experimental model of post-viral lung disease that requires basal-epithelial stem cell growth, immune activation, and differentiation. The present results thereby provide evidence of possible basal epithelial cell reprogramming in long-term Covid-19 as well and thereby a pathway for explaining and correcting lung dysfunction in this type of disease.

Viral Evasion of Innate Immune Defense: The Case of Resistance of Pandemic H1N1 Influenza A Virus to Human Mannose-Binding Proteins.

Mannose-binding lectins effectively inhibit most seasonal strains of influenza A virus and contribute to the innate host defense vs. these viruses. In contrast, pandemic IAV strains are largely resistant to these lectins, likely contributing to increased spread and worse outcomes. In this paper, we evaluated the inhibition of IAV by mannose-binding lectins of human, bacterial, and fungal origin to understand and possibly increase activity vs. the pandemic IAV. A modified version of the human surfactant protein D (SP-D) neck and carbohydrate recognition domain (NCRD) with combinatorial substitutions at the 325 and 343 positions, previously shown to inhibit pandemic H3N2 IAV in vitro and in vivo, and to inhibit pandemic H1N1 in vitro, failed to protect mice from pandemic H1N1 in vivo in the current study. We attempted a variety of maneuvers to improve the activity of the mutant NCRDs vs. the 2009 pandemic H1N1, including the formation of full-length SP-D molecules containing the mutant NCRD, cross-linking of NCRDs through the use of antibodies, combining SP-D or NCRDs with alpha-2-macroglobulin, and introducing an additional mutation to the double mutant NCRD. None of these substantially increased the antiviral activity for the pandemic H1N1. We also tested the activity of bacterial and algal mannose-binding lectins, cyanovirin, and griffithsin, against IAV. These had strong activity against seasonal IAV, which was largely retained against pandemic H1N1. We propose mechanisms to account for differences in activity of SP-D constructs against pandemic H3N2 and H1N1, and for differences in activity of cyanovirin vs. SP-D constructs.

Basal epithelial stem cells cross an alarmin checkpoint for postviral lung disease.

Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease.

Collectins, H-ficolin and LL-37 reduce influence viral replication in human monocytes and modulate virus-induced cytokine production.

Infiltrating activated monocytes are important mediators of damaging inflammation during influenza A virus (IAV) infection. We show that soluble respiratory proteins [collectins, surfactant proteins D (SP-D) and mannose binding lectin (MBL), H-ficolin and LL-37] inhibit replication of seasonal IAV in human monocytes. The collectins and H-ficolin also increased viral uptake by the cells, while LL-37 did not. H-ficolin was able to inhibit replication of the 2009 pandemic H1N1 strain (Cal09) in monocytes, but SP-D and LL-37 had significantly fewer inhibitory effects on this strain than on seasonal IAV. All of these proteins reduced IAV-induced TNF-α production, even in instances when viral replication was not reduced. We used modified recombinant versions of SP-D, MBL and ficolin to elucidate mechanisms through which these proteins alter monocyte interactions with IAV. We demonstrate the importance of the multimeric structure, and of binding properties of the lectin domain, in mediating antiviral and opsonic activity of the proteins. Hence, soluble inhibitors present in airway lining fluid may aid clearance of IAV by promoting monocyte uptake of the virus, while reducing viral replication and virus-induced TNF-α responses in these cells. However, SP-D and LL-37 have reduced ability to inhibit replication of pandemic IAV in monocytes.

Structure binding relationship of human surfactant protein D and various lipopolysaccharide inner core structures.

As a major player of the innate immune system, surfactant protein D (SP-D) recognizes and promotes elimination of various pathogens such as Gram-negative bacteria. SP-D binds to l-glycero-d-manno-heptose (Hep), a constituent of the partially conserved lipopolysaccharide (LPS) inner core of many Gram-negative bacteria. Binding and affinity of trimeric human SP-D to Hep in distinct LPS inner core glycans differing in linkages and adjacent residues was elucidated using glycan array and surface plasmon resonance measurements that were compared to in silico interaction studies. The combination of in vitro assays using defined glycans and molecular docking and dynamic simulation approaches provides insights into the interaction of trimeric SP-D with those glycan ligands. Trimeric SP-D wildtype recognized larger LPS inner core oligosaccharides with slightly enhanced affinity than smaller compounds suggesting the involvement of stabilizing secondary interactions. A trimeric human SP-D mutant D324N+D325N+R343K resembling rat SP-D bound to various LPS inner core structures in a similar pattern as observed for the wildtype but with higher affinity. The selective mutation of SP-D promotes targeting of LPS inner core oligosaccharides on Gram-negative bacteria to develop novel therapeutic agents.

Computational Analysis Reveals a Key Regulator of Cryptococcal Virulence and Determinant of Host Response.

UNLABELLED: Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills over 600,000 people annually. Here, we report integrated computational and experimental investigations of the role and mechanisms of transcriptional regulation in cryptococcal infection. Major cryptococcal virulence traits include melanin production and the development of a large polysaccharide capsule upon host entry; shed capsule polysaccharides also impair host defenses. We found that both transcription and translation are required for capsule growth and that Usv101 is a master regulator of pathogenesis, regulating melanin production, capsule growth, and capsule shedding. It does this by directly regulating genes encoding glycoactive enzymes and genes encoding three other transcription factors that are essential for capsule growth: GAT201, RIM101, and SP1. Murine infection with cryptococci lacking Usv101 significantly alters the kinetics and pathogenesis of disease, with extended survival and, unexpectedly, death by pneumonia rather than meningitis. Our approaches and findings will inform studies of other pathogenic microbes. IMPORTANCE: Cryptococcus neoformans causes fatal meningitis in immunocompromised individuals, mainly HIV positive, killing over 600,000 each year. A unique feature of this yeast, which makes it particularly virulent, is its polysaccharide capsule; this structure impedes host efforts to combat infection. Capsule size and structure respond to environmental conditions, such as those encountered in an infected host. We have combined computational and experimental tools to elucidate capsule regulation, which we show primarily occurs at the transcriptional level. We also demonstrate that loss of a novel transcription factor alters virulence factor expression and host cell interactions, changing the lethal condition from meningitis to pneumonia with an exacerbated host response. We further demonstrate the relevant targets of regulation and kinetically map key regulatory and host interactions. Our work elucidates mechanisms of capsule regulation, provides methods and resources to the research community, and demonstrates an altered pathogenic outcome that resembles some human conditions.

Homeostatic Control of Innate Lung Inflammation by Vici Syndrome Gene Epg5 and Additional Autophagy Genes Promotes Influenza Pathogenesis.

Mutations in the autophagy gene EPG5 are linked to the multisystem human disease Vici syndrome, which is characterized in part by pulmonary abnormalities, including recurrent infections. We found that Epg5-deficient mice exhibited elevated baseline innate immune cellular and cytokine-based lung inflammation and were resistant to lethal influenza virus infection. Lung transcriptomics, bone marrow transplantation experiments, and analysis of cellular cytokine expression indicated that Epg5 plays a role in lung physiology through its function in macrophages. Deletion of other autophagy genes including Atg14, Fip200, Atg5, and Atg7 in myeloid cells also led to elevated basal lung inflammation and influenza resistance. This suggests that Epg5 and other Atg genes function in macrophages to limit innate immune inflammation in the lung. Disruption of this normal homeostatic dampening of lung inflammation results in increased resistance to influenza, suggesting that normal homeostatic mechanisms that limit basal tissue inflammation support some infectious diseases.

Mutations flanking the carbohydrate binding site of surfactant protein D confer antiviral activity for pandemic influenza A viruses.

We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan-rich viral envelope proteins including hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or increased neutralizing activity compared with D325A+R343V. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild-type SP-D NCRD did not. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan side on the head region of the HA and are fully resistant to inhibition by native SP-D. Importantly, we now show that D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains, and reduced uptake of Cal09 by epithelial cells. Inhibition of Cal09 was mediated by the lectin activity of the NCRDs. All known human pandemic strains have at least one glycan attachment on the top or side of the HA head, and our results indicate that they may be susceptible to inhibition by modified host defense lectins.

Molecular mechanisms of inhibition of influenza by surfactant protein D revealed by large-scale molecular dynamics simulation.

Surfactant protein D (SP-D), a mammalian C-type lectin, is the primary innate inhibitor of influenza A virus (IAV) in the lung. Interactions of SP-D with highly branched viral N-linked glycans on hemagglutinin (HA), an abundant IAV envelope protein and critical virulence factor, promote viral aggregation and neutralization through as yet unknown molecular mechanisms. Two truncated human SP-D forms, wild-type (WT) and double mutant D325A+R343V, representing neck and carbohydrate recognition domains are compared in this study. Whereas both WT and D325A+R343V bind to isolated glycosylated HA, WT does not inhibit IAV in neutralization assays; in contrast, D325A+R343V neutralization compares well with that of full-length native SP-D. To elucidate the mechanism for these biochemical observations, we have determined crystal structures of D325A+R343V in the presence and absence of a viral nonamannoside (Man9). On the basis of the D325A+R343V-Man9 structure and other crystallographic data, models of complexes between HA and WT or D325A+R343V were produced and subjected to molecular dynamics. Simulations reveal that whereas WT and D325A+R343V both block the sialic acid receptor site of HA, the D325A+R343V complex is more stable, with stronger binding caused by additional hydrogen bonds and hydrophobic interactions with HA residues. Furthermore, the blocking mechanism of HA differs for WT and D325A+R343V because of alternate glycan binding modes. The combined results suggest a mechanism through which the mode of SP-D-HA interaction could significantly influence viral aggregation and neutralization. These studies provide the first atomic-level molecular view of an innate host defense lectin inhibiting its viral glycoprotein target.

Increased surfactant protein D fails to improve bacterial clearance and inflammation in serpinB1-/- mice.

Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis.

A unique sugar-binding site mediates the distinct anti-influenza activity of pig surfactant protein D.

Pigs can act as intermediate hosts by which reassorted influenza A virus (IAV) strains can be transmitted to humans and cause pandemic influenza outbreaks. The innate host defense component surfactant protein D (SP-D) interacts with glycans on the hemagglutinin of IAV and contributes to protection against IAV infection in mammals. This study shows that a recombinant trimeric neck lectin fragment derived from porcine SP-D (pSP-D) exhibits profound inhibitory activity against IAV, in contrast to comparable fragments derived from human SP-D. Crystallographic analysis of the pSP-D fragment complexed with a viral sugar component shows that a unique tripeptide loop alters the lectin site conformation of pSP-D. Molecular dynamics simulations highlight the role of this flexible loop, which adopts a more stable conformation upon sugar binding and may facilitate binding to viral glycans through contact with distal portions of the branched mannoside. The combined data demonstrate that porcine-specific structural features of SP-D contribute significantly to its distinct anti-IAV activity. These findings could help explain why pigs serve as important reservoirs for newly emerging pathogenic IAV strains.

Hapivirins and diprovirins: novel θ-defensin analogs with potent activity against influenza A virus.

θ-Defensins are cyclic octadecapeptides found in nonhuman primates whose broad antiviral spectrum includes HIV-1, HSV-1, severe acute respiratory syndrome coronavirus, and influenza A virus (IAV). We previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This study describes two families of peptides, hapivirins and diprovirins, whose design was inspired by retrocyclins. The goal was to develop smaller partially cyclic peptides that retain the antiviral activity of retrocyclins, while being easier to synthesize. The novel peptides also allowed for systemic substitution of key residues to evaluate the role of charge or hydrophobicity on antiviral activity. Seventy-two hapivirin or diprovirin peptides are described in this work, including several whose anti-IAV activity equals or exceeds that of normal α- or θ-defensins. Some of these also had strong antibacterial and antifungal activity. These new peptides were active against H3N2 and H1N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum t(1/2) in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection, and, when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogs provides an approach for creating novel antiviral agents for IAV infections.

The ability of pandemic influenza virus hemagglutinins to induce lower respiratory pathology is associated with decreased surfactant protein D binding.

Pandemic influenza viral infections have been associated with viral pneumonia. Chimeric influenza viruses with the hemagglutinin segment of the 1918, 1957, 1968, or 2009 pandemic influenza viruses in the context of a seasonal H1N1 influenza genome were constructed to analyze the role of hemagglutinin (HA) in pathogenesis and cell tropism in a mouse model. We also explored whether there was an association between the ability of lung surfactant protein D (SP-D) to bind to the HA and the ability of the corresponding chimeric virus to infect bronchiolar and alveolar epithelial cells of the lower respiratory tract. Viruses expressing the hemagglutinin of pandemic viruses were associated with significant pathology in the lower respiratory tract, including acute inflammation, and showed low binding activity for SP-D. In contrast, the virus expressing the HA of a seasonal influenza strain induced only mild disease with little lung pathology in infected mice and exhibited strong in vitro binding to SP-D.

Pandemic H1N1 influenza A viruses are resistant to the antiviral activities of innate immune proteins of the collectin and pentraxin superfamilies.

Acquired immune responses elicited to recent strains of seasonal H1N1 influenza viruses provide limited protection against emerging A(H1N1) pandemic viruses. Accordingly, pre-existing or rapidly induced innate immune defenses are of critical importance in limiting early infection. Respiratory secretions contain proteins of the innate immune system, including members of the collectin and pentraxin superfamilies. These mediate potent antiviral activity and act as an initial barrier to influenza infection. In this study, we have examined the sensitivity of H1N1 viruses, including pandemic virus strains, for their sensitivity to collectins (surfactant protein [SP]-D and mannose-binding lectin [MBL]) and to the pentraxin PTX3. Human SP-D and MBL inhibited virus-induced hemagglutinating activity, blocked the enzymatic activity of the viral neuraminidase, and neutralized the ability of H1N1 viruses to infect human respiratory epithelial cells in a manner that correlated with the degree of glycosylation in the globular head of the hemagglutinin. Recent seasonal H1N1 viruses expressed three to four N-glycosylation sequons on the head of hemagglutinin and were very sensitive to inhibition by SP-D or MBL, whereas A(H1N1) pandemic viruses expressed a single N-glycosylation sequon and were resistant to either collectin. Of interest, both seasonal and pandemic H1N1 viruses were resistant to PTX3. Thus, unlike recent seasonal H1N1 strains of influenza virus, A(H1N1) pandemic viruses are resistant to the antiviral activities of innate immune proteins of the collectin superfamily.

Monoclonal antibody-assisted structure-function analysis of the carbohydrate recognition domain of surfactant protein D.

Surfactant protein D (SP-D) plays important roles in host defense against a variety of pathogens including influenza A virus (IAV). Ligand binding by SP-D is mediated by the trimeric neck and carbohydrate recognition domain (NCRD). We used monoclonal antibodies (mAbs) against human SP-D and a panel of mutant collectin NCRD constructs to identify functionally and structurally important epitopes. The ability of SP-D to bind to IAV and mannan involved partially overlapping binding sites that are distinct from those involved in binding to the glycoprotein-340 (gp-340) scavenger receptor protein. A species-specific motif (D324,D325,R343), which has been implicated in the specific binding of several ligands, contributes to recognition by mAbs that block antiviral or mannan binding activity. D325, in particular, is involved in the epitopes of these blocking mAbs. Conversely, the interspecies substitution of arginine for Lys343 in the rat NCRD (rK343R) conferred binding to two of the mAbs. The single site substitution of alanine for R349 or E347 resulted in highly selective alterations in mAb binding and caused decreased antiviral activity. Mutations at Glu333 (E333A), Trp340 (W340F), and Phe335 (F335A), which abrogated antiviral activity, were associated with decreased binding to multiple blocking mAbs, consistent with critical structural roles. More conservative substitutions at 335, which showed a significant increase in neutralization activity, caused selective loss of binding to one mAb. The analysis reveals, for the first time, an extended binding site for IAV; calcium-dependent antiviral activity involves residues flanking the primary carbohydrate binding site as well as more remote residues displayed on the carbohydrate recognition domain surface.

Surfactant protein D-mediated decrease of allergen-induced inflammation is dependent upon CTLA4.

Pulmonary surfactant protein D (SP-D), a member of the collectin family, is an innate immune molecule critical for defense that can also modulate adaptive immune responses. We previously showed that SP-D-deficient mice exhibit enhanced allergic responses and that SP-D induction requires lymphocytes. Thus, we postulated that SP-D may decrease adaptive allergic responses through interaction with T cells. In this study, we used two forms of SP-D, a dodecamer and a shorter fragment containing the trimeric neck and carbohydrate recognition domains (SP-D NCRD). Both forms decreased immune responses in vitro and in a murine model of pulmonary inflammation. SP-D NCRD increased transcription of CTLA4, a negative regulator of T cell activation, in T cells. SP-D NCRD no longer decreased lymphoproliferation and IL-2 cytokine production when CTLA4 signals were abrogated. Administration of SP-D NCRD in vivo no longer decreased allergen induced responses when CTLA4 was inhibited. Our results indicate that SP-D decreases allergen responses, an effect that may be mediated by increase of CTLA4 in T cells.

Review: Structural determinants of pattern recognition by lung collectins.

Host defense roles for the lung collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), were first suspected in the 1980s when molecular characterization revealed their sequence homology to the acute phase reactant of serum, mannose-binding lectin. Surfactant protein A and SP-D have since been shown to play diverse and important roles in innate immunity and pulmonary homeostasis. Their location in surfactant ideally positions them to interact with air-space pathogens. Despite extensive structural similarity, the two proteins show many functional differences and considerable divergence in their interactions with microbial surface components, surfactant lipids, and other ligands. Recent crystallographic studies have provided many new insights relating to these observed differences. Although both proteins can participate in calcium-dependent interactions with sugars and other polyols, they display significant differences in the spatial orientation, charge, and hydrophobicity of their binding surfaces. Surfactant protein D appears particularly adapted to interactions with complex carbohydrates and anionic phospholipids, such as phosphatidylinositol. By contrast, SP-A shows features consistent with its preference for lipid ligands, including lipid A and the major surfactant lipid, dipalmitoylphosphatidylcholine. Current research suggests that structural biology approaches will help to elucidate the molecular basis of pulmonary collectin-ligand recognition and facilitate development of new therapeutics based upon SP-A and SP-D.

Enhancement of antiviral activity of collectin trimers through cross-linking and mutagenesis of the carbohydrate recognition domain.

Surfactant protein D (SP-D) plays important roles in innate defense against respiratory viruses [including influenza A viruses (IAVs)]. Truncated trimers composed of its neck and carbohydrate recognition domains (NCRDs) bind various ligands; however, they have minimal inhibitory activity for IAV. We have sought to find ways to increase the antiviral activity of collectin NCRDs. Cross-linking of the SP-D NCRD with nonblocking monoclonal antibodies (mAbs) markedly potentiates antiviral activity. In the present report, we demonstrate that F(ab')2 [but not F(ab')1] fragments of a cross-linking mAb have similar effects. Hence, cross-linking activity, but not the Fc domain of the mAb, is needed for increased antiviral activity. In contrast, the Fc domain of the mAb was important for increasing viral uptake or respiratory burst responses of human neutrophils. Our NCRD constructs contain an S protein binding site. Herein, we show that a multivalent S protein complex caused cross-linking and also increased the antiviral activity of NCRDs. NCRDs of conglutinin and CL43 had greater intrinsic antiviral activity than those of SP-D or mannose-binding lectin. Based on motifs found in these serum collectins, we have constructed mutant versions of the human SP-D NCRD that have increased antiviral activity. These mutant NCRDs also had potentiated activity after cross-linking with F(ab')2 fragments or S protein complexes. Hence, the antiviral activity of NCRDs can be increased by 2 distinct, complementary strategies, namely cross-linking of NCRDs through various means and mutagenesis of CRD residues to increase viral binding. These findings may be relevant for antiviral therapy.

Myeloperoxidase-dependent inactivation of surfactant protein D in vitro and in vivo.

Surfactant protein D (SP-D) plays diverse and important roles in innate immunity and pulmonary homeostasis. Neutrophils and myeloperoxidase (MPO) colocalized with SP-D in a murine bacterial pneumonia model of acute inflammation, suggesting that MPO-derived reactive species might alter the function of SP-D. Exposure of SP-D to the complete MPO-H(2)O(2)-halide system caused loss of SP-D-dependent aggregating activity. Hypochlorous acid (HOCl), the major oxidant generated by MPO, caused a similar loss of aggregating activity, which was accompanied by the generation of abnormal disulfide-cross-linked oligomers. A full-length SP-D mutant lacking N-terminal cysteine residues and truncation mutants lacking the N-terminal domains were resistant to the oxidant-induced alterations in disulfide bonding. Mass spectroscopy of HOCl-treated human SP-D demonstrated several modifications, but none involved key ligand binding residues. There was detectable oxidation of cysteine 15, but no HOCl-induced cysteine modifications were observed in the C-terminal lectin domain. Together, the findings localize abnormal disulfide cross-links to the N-terminal domain. MPO-deficient mice showed decreased cross-linking of SP-D and increased SP-D-dependent aggregating activity in the pneumonia model. Thus, MPO-derived oxidants can lead to modifications of SP-D structure with associated alterations in its characteristic aggregating activity.