Dysregulated serum and seminal plasma levels of surfactant protein D and MCP-1 in men with genital tract infection/inflammation.

PROBLEM: Surfactant protein D (SP-D), a multimeric collectin expressed by testicular mucosal epithelia and is positively regulated by testosterone. It exerts antimicrobial effects, modulates inflammation and rescued spermatogenesis in a murine model. Various cytokines and chemokines, including MCP-1, play a key role in regulating the inflammation in rat and human testis. The study aimed to investigate the role of SP-D and involvement of chemokines and cytokines in the male infertility associated with urogenital infections or inflammation. METHOD OF STUDY: The cross-sectional study evaluated levels of SP-D, testosterone, estradiol and the cytokines/chemokines including MCP-1 in the serum and semen samples of fertile and infertile Indian men with and without urogenital infections/inflammation (n = 76). RESULTS: Both fertile and infertile males with urogenital infection/inflammation had significantly lower levels of SP-D and higher levels of the chemokine, Monocyte chemoattractant protein 1 (MCP-1) in the serum and seminal plasma. Seminal plasma of these males exhibited significantly higher proportion of proteolytically degraded forms of SP-D. The serum SP-D levels positively correlated with testosterone/estradiol (TE) ratio. There was no significant correlation between the SP-D levels in seminal plasma and sperm count/motility. With a significant area under the Receiver Operating Characteristic curves, the serum and seminal plasma SP-D levels exhibited significant potential to predict infertility with high sensitivity and specificity in men with genital infections/inflammation. CONCLUSIONS: The circulating and seminal plasma SP-D levels were decreased in men with urogenital infection and inflammation. This could be due to their engagement at the site of infection, dysregulated expression owing to the altered hormonal profile and increased proteolytic degradation.

Immunomodulatory Role of Surfactant Protein-D in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) Model.

Surfactant protein D (SP-D), a pattern recognition molecule, is emerging as a potent anti-tumoural innate immune defense molecule in a range of cancers. Previously, SP-D expression was found to be significantly downregulated at the malignant sites of human prostate adenocarcinoma and associated with an increasing Gleason score and severity. We recently reported selective induction of intrinsic apoptosis by a recombinant fragment of human SP-D (rfhSP-D) in the human Prostate cancer (PCa) biopsy explants and cells with glucose regulated protein of 78 (GRP78) as one of the key interacting partners. The present study evaluated the expression of SP-D in early and advanced stages of PCa using transgenic adenocarcinoma of mouse prostate (TRAMP) model. Both early and late stages of PCa showed significantly decreased SP-D mRNA expression and increased proteolytic degradation of SP-D protein. Systemic and tumoural immunophenotyping of TRAMP model revealed increased serine proteases producing granulocytes and polymorphonuclear myeloid-derived suppressor cells (PMN MDSCs) in the late stage; the serine proteases secreted by these cells could be involved in the degradation of SP-D. Susceptibility of rfhSP-D to elastase-mediated proteolysis provided the rationale to use an elastase-inhibitor to sustain intact rfhSP-D in the tumour microenvironment. The study revealed an immunomodulatory potential of rfhSP-D and elastase inhibitor, sivelestat, to induce macrophage polarization towards M1 with downregulation of PMN MDSCs in ex-vivo cultured TRAMP tumours. Furthermore, rfhSP-D induced immunogenic cell death in murine PCa cells and in TRAMP explants. The findings highlight that SP-D plays an anti-tumourigenic role in PCa by inducing immunogenic cell death and immunomodulation while the prostate tumour milieu adversely impacts SP-D by inhibiting its transcription, and enhancing its proteolytic degradation. Transformation of an immunologically "cold tumour" into a "hot tumour" implicates therapeutic potential of rfhSP-D in PCa.

Interplay between C-type lectin receptors and microRNAs in cellular homeostasis and immune response.

C-type lectin receptors (CLRs) belong to the family of pattern recognition receptors (PRRs). They have a critical role to play in the regulation of a range of physiological functions including development, respiration, angiogenesis, inflammation, and immunity. CLRs can recognize distinct and conserved exogenous pathogen-associated as well as endogenous damage-associated molecular patterns. These interactions set off downstream signaling cascades, leading to the production of inflammatory mediators, activation of effector immune cells as well as regulation of the developmental and physiological homeostasis. CLR signaling must be tightly controlled to circumvent the excessive inflammatory burden and to maintain the cellular homeostasis. Recently, MicroRNAs (miRNAs) have been shown to be important regulators of expression of CLRs and their downstream signaling. The delicate balance between miRNAs and CLRs seems crucial in almost all aspects of multicellular life. Any dysregulations in the miRNA-CLR axes may lead to tumorigenesis or inflammatory diseases. Here, we present an overview of the current understanding of the central role of miRNAs in the regulation of CLR expression, profoundly impacting upon homeostasis and immunity, and thus, development of therapeutics against immune disorders.

Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells.

Human surfactant protein D (SP-D) belongs to the family of collectins that is composed of a characteristic amino-terminal collagenous region and a carboxy-terminal C-type lectin domain. Being present at the mucosal surfaces, SP-D acts as a potent innate immune molecule and offers protection against non-self and altered self, such as pathogens, allergens, and tumor. Here, we examined the effect of a recombinant fragment of human SP-D (rfhSP-D) on a range of breast cancer lines. Breast cancer has four molecular subtypes characterized by varied expressions of estrogen (ER), progesterone (PR), and epidermal growth factor (EGF) receptors (HER2). The cell viability of HER2-overexpressing (SKBR3) and triple-positive (BT474) breast cancer cell lines [but not of a triple-negative cell line (BT20)] was reduced following rfhSP-D treatment at 24 h. Upregulation of p21/p27 cell cycle inhibitors and p53 phosphorylation (Ser15) in rfhSP-D-treated BT474 and SKBR3 cell lines signified G2/M cell cycle arrest. Cleaved caspases 9 and 3 were detected in rfhSP-D-treated BT474 and SKBR3 cells, suggesting an involvement of the intrinsic apoptosis pathway. However, rfhSP-D-induced apoptosis was nullified in the presence of hyaluronic acid (HA) whose increased level in breast tumor microenvironment is associated with malignant tumor progression and invasion. rfhSP-D bound to solid-phase HA and promoted tumor cell proliferation. rfhSP-D-treated SKBR3 cells in the presence of HA showed decreased transcriptional levels of p53 when compared to cells treated with rfhSP-D only. Thus, HA appears to negate the anti-tumorigenic properties of rfhSP-D against HER2-overexpressing and triple-positive breast cancer cells.

Differential Interactions of Serum and Bronchoalveolar Lavage Fluid Complement Proteins with Conidia of Airborne Fungal Pathogen Aspergillus fumigatus.

Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, ß-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche.

Membrane Interactome of a Recombinant Fragment of Human Surfactant Protein D Reveals GRP78 as a Novel Binding Partner in PC3, a Metastatic Prostate Cancer Cell Line.

Surfactant protein-D (SP-D), a member of the collectin family has been shown to induce apoptosis in cancer cells. SP-D is composed of an N-terminal collagen-like domain and a calcium-dependent carbohydrate recognition domain (CRD). Recently, we reported that a recombinant fragment of human SP-D (rfhSP-D), composed of homotrimeric CRD region, induced intrinsic apoptotic pathway in prostate cancer cells. Here, we analyzed the membrane interactome of rfhSP-D in an androgen-independent prostate cancer cell line, PC3, by high resolution mass spectrometry and identified 347 proteins. Computational analysis of PPI network of this interactome in the context of prostate cancer metastasis and apoptosis revealed Glucose Regulated Protein of 78 kDa (GRP78) as an important binding partner of rfhSP-D. Docking studies suggested that rfhSP-D (CRD) bound to the substrate-binding domain of glycosylated GRP78. This was further supported by the observations that human recombinant GRP78 interfered with the binding of rfhSP-D to anti-SP-D polyclonal antibodies; GRP78 also significantly inhibited the binding of recombinant full-length human SP-D with a monoclonal antibody specific to the CRD in a dose-dependent manner. We conclude that the interaction with rfhSP-D is likely to interfere with the pro-survival signaling of GRP78.

Differential levels of surfactant protein A, surfactant protein D, and progesterone to estradiol ratio in maternal serum before and after the onset of severe early-onset preeclampsia.

PROBLEM: Preeclampsia (PE), a multifactorial disorder characterized by impaired placental development, elevated inflammatory response and dysregulated placental steroidogenesis. PE may be preventable if predicted early on. METHOD OF STUDY: The study evaluated the potential of immunomodulatory collectins, surfactant protein A (SP-A), surfactant protein D (SP-D), and mannose binding lectin (MBL), to predict PE before the disease onset, in a prospective study cohort of healthy pregnant women (n = 922). In addition, a cross-sectional study was conducted to determine the serum and placental profile of collectins in PE women after the disease onset (early-onset PE [EOPE], n = 33; late-onset PE [LOPE], n = 24); and controls [n = 75]. The serum profiles of estradiol (E2) and progesterone (P4) were evaluated to determine their correlation with collectins. RESULTS: In the prospective cohort, significantly decreased serum levels of SP-A, SP-D, P4/E2 ratio were observed in women who subsequently developed severe EOPE. Interestingly, after the disease onset, there was a significant increase in serum and placental levels of collectins in women with severe EOPE, whereas women with LOPE had significantly decreased levels of collectins. Serum P4/E2 ratio was significantly altered in severe EOPE and positively correlated with serum levels of SP-A and SP-D. CONCLUSION: Collectins are differentially expressed in the serum during progression of PE. Decreased serum levels of SP-A, SP-D, P4/E2 ratio and increased E2 during 10-20 weeks of gestation are novel plausible risk factors for early prediction of EOPE in Indian women.

Human SP-D Acts as an Innate Immune Surveillance Molecule Against Androgen-Responsive and Androgen-Resistant Prostate Cancer Cells.

Surfactant Protein D (SP-D), a pattern recognition innate immune molecule, has been implicated in the immune surveillance against cancer. A recent report showed an association of decreased SP-D expression in human prostate adenocarcinoma with an increased Gleason score and severity. In the present study, the SP-D expression was evaluated in primary prostate epithelial cells (PrEC) and prostate cancer cell lines. LNCaP, an androgen dependent prostate cancer cell line, exhibited significantly lower mRNA and protein levels of SP-D than PrEC and the androgen independent cell lines (PC3 and DU145). A recombinant fragment of human SP-D, rfhSP-D, showed a dose and time dependent binding to prostate cancer cells via its carbohydrate recognition domain. This study, for the first time, provides evidence of significant and specific cell death of tumor cells in rfhSP-D treated explants as well as primary tumor cells isolated from tissue biopsies of metatstatic prostate cancer patients. Viability of PrEC was not altered by rfhSP-D. Treated LNCaP (p53+/+) and PC3 (p53 -/-) cells exhibited reduced cell viability in a dose and time dependent manner and were arrested in G2/M and G1/G0 phase of the cell cycle, respectively. rfhSP-D treated LNCaP cells showed a significant upregulation of p53 whereas a significant downregulation of pAkt was observed in both PC3 and LNCaP cell lines. The rfhSP-D-induced apoptosis signaling cascade involved upregulation of Bax:Bcl2 ratio, cytochrome c and cleaved products of caspase 7. The study concludes that rfhSP-D induces apoptosis in prostate tumor explants as well as in androgen dependent and independent prostate cancer cells via p53 and pAkt pathways.

Surfactant Protein D Reverses the Gene Signature of Transepithelial HIV-1 Passage and Restricts the Viral Transfer Across the Vaginal Barrier.

Effective prophylactic strategy against the current epidemic of sexually transmitted HIV-1 infection requires understanding of the innate gatekeeping mechanisms at the genital mucosa. Surfactant protein D (SP-D), a member of the collectin family of proteins naturally present in the vaginal tract, is a potential HIV-1 entry inhibitor at the cellular level. Human EpiVaginal tissues compartmentalized in culture inserts were apically exposed to HIV-1 and/or a recombinant fragment of human SP-D (rfhSP-D) and viral passage was assessed in the basal chamber containing mononuclear leukocytes. To map the gene signature facilitating or resisting the transepithelial viral transfer, microarray analysis of the HIV-1 challenged EpiVaginal tissues was performed in the absence or presence of rfhSP-D. Mucosal biocompatibility of rfhSP-D was assessed ex vivo and in the standard rabbit vaginal irritation model. The passage of virus through the EpiVaginal tissues toward the underlying target cells was associated with a global epithelial gene signature including differential regulation of genes primarily involved in inflammation, tight junctions and cytoskeletal framework. RfhSP-D significantly inhibited HIV-1 transfer across the vaginal tissues and was associated with a significant reversal of virus induced epithelial gene signature. Pro-inflammatory NF-κB and mTOR transcripts were significantly downregulated, while expression of the tight junctions and cytoskeletal genes was upheld. In the absence of virus, rfhSP-D directly interacted with the EpiVaginal tissues and upregulated expression of genes related to structural stability of the cell and epithelial integrity. There was no increment in the viral acquisition by the PBMCs present in basal chambers wherein, the EpiVaginal tissues in apical chambers were treated with rfhSP-D. The effective concentrations of rfhSP-D had no effect on lactobacilli, epithelial barrier integrity and were safe on repeated applications onto the rabbit vaginal mucosa. This pre-clinical safety data, coupled with its efficacy of restricting viral passage via reversal of virus-induced gene expression of the vaginal barrier, make a strong argument for clinical trials of rfhSP-D as a topical anti-HIV microbicide.

Aspergillus fumigatus conidial metalloprotease Mep1p cleaves host complement proteins.

Innate immunity in animals including humans encompasses the complement system, which is considered an important host defense mechanism against Aspergillus fumigatus, one of the most ubiquitous opportunistic human fungal pathogens. Previously, it has been shown that the alkaline protease Alp1p secreted from A. fumigatus mycelia degrades the complement components C3, C4, and C5. However, it remains unclear how the fungal spores (i.e. conidia) defend themselves against the activities of the complement system immediately after inhalation into the lung. Here, we show that A. fumigatus conidia contain a metalloprotease Mep1p, which is released upon conidial contact with collagen and inactivates all three complement pathways. In particular, Mep1p efficiently inactivated the major complement components C3, C4, and C5 and their activation products (C3a, C4a, and C5a) as well as the pattern-recognition molecules MBL and ficolin-1, either by directly cleaving them or by cleaving them to a form that is further broken down by other proteases of the complement system. Moreover, incubation of Mep1p with human serum significantly inhibited the complement hemolytic activity and conidial opsonization by C3b and their subsequent phagocytosis by macrophages. Together, these results indicate that Mep1p associated with and released from A. fumigatus conidia likely facilitates early immune evasion by disarming the complement defense in the human host.

Estradiol: A Steroid with Multiple Facets.

Seventy-five glorious years have passed since estradiol was discovered by Edward Doisy. From discovery in the ovaries to delineation of diverse physiological effects, research on estrogens has covered a lot of ground. Estrogen receptors that mediate estrogenic effects, have been detected not only in reproductive organs, but also in other body organs. Estrogen receptors function either as conventional transcription factors or as rapid signal transducers. These different modes of action are opted by estrogens to elicit an array of reproductive and non-reproductive functions. It is well established that estrogens promote cell proliferation in various tissues and hence are also linked to carcinogenesis. Anti-estrogens are being used as adjunct therapies for cancers since several years. On the other hand, estrogen-based strategies are used to alleviate adverse effects of menopause. Apart from estrogens synthesized in various organs, exposure to environmental estrogens can also impact physiology. Thus, too much or too less of estrogens can tip the balance and lead to unfavorable consequences. Multiple estrogen receptors with their tissue- or cell type-specific expression eliciting dose-dependent effects make it perplexing to 'unify' estrogenic actions in diverse tissues/organs. This warrants more research on estrogen-mediated effects and their regulation in somatic and reproductive tissues. This review presents physiological and pathological aspects of estrogens thus highlighting the good, bad, and ugly facets of estrogens.

Mesenchymal stem cells: a promising tool for targeted gene therapy of endometriosis.

Endometriosis is a leading, benign gynecological disorder around the world. Last few years have witnessed tremendous growth in the field of endometriosis and endometrial stem-cell research. Despite advancements in the biology and pathology of endometriosis, disease recurrence is still an enigma. Gene therapy holds promise in treating many pathologic conditions including endometriosis. Mesenchymal stem cells (MSCs) serve as ideal candidates for regenerative medicine and cell-based therapies. Owing to their specificity to the endometrium, residing endometrial MSC populations could be utilized as ideal candidates for targeting endometrial disorders. Recently, we demonstrated their flexibility for gene transduction using adenoviral vectors. The review highlights the potential of endometrial MSCs in devising targeted gene therapies for endometriosis.

Expression of surfactant proteins SP-A and SP-D in murine decidua and immunomodulatory effects on decidual macrophages.

Surfactant proteins SP-A and SP-D are pattern recognition innate immune molecules that belong to the C-type lectin family. In lungs, they play an important role in the clearance of pathogens and control of inflammation. SP-A and SP-D are also expressed in the female reproductive tract where they play an important role in pregnancy and parturition. However, the role of SP-A and SP-D expressed at the feto-maternal interface (decidua) remains unclear. Here, we have examined the expression of SP-A and SP-D in the murine decidua at 17.5 (pre-parturition) and 19.5dpc (near parturition) and their effect on lipopolysaccharide (LPS)-treated decidual macrophages. SP-A and SP-D were localized to stromal cells in the murine decidua at 17.5 and 19.5dpc in addition to cells lining the maternal spiral artery. Purified pre-parturition decidual cells were challenged with LPS with and without SP-A or SP-D, and expression of F4/80 and TNF-α were measured by flow cytometry. On their own, SP-A or SP-D did not affect the percentage of F4/80 positive cells while they suppressed the percentage of TNF-α positive cells. However, simultaneous addition of SP-A or SP-D, together with LPS, reduced TNF-α secreting F4/80 positive cells. It is likely that exogenous administration of SP-A and SP-D in decidua can potentially control infection and inflammation mediators during spontaneous term labor and infection-induced preterm labor. Thus, the presence of SP-A and SP-D in the murine decidua is likely to play a protective role against intrauterine infection during pregnancy.

Surfactant protein D induces immune quiescence and apoptosis of mitogen-activated peripheral blood mononuclear cells.

Surfactant protein D (SP-D) is an integral molecule of the innate immunity secreted by epithelial cells lining the mucosal surfaces. The C-type lectin domain of SP-D performs pattern recognition functions while it binds to putative receptors on immune cells to modify cellular functions. Activation of immune cells and increased serum SP-D is observed in a range of patho-physiological conditions including infections. We speculated if SP-D can modulate systemic immune response via direct interaction with activated PBMCs. In this study, we examined interaction of a recombinant fragment of human SP-D (rhSP-D) on PHA-activated PBMCs. We report a significant downregulation of activation receptors such as TLR2, TLR4, CD11c and CD69 upon rhSP-D treatment. rhSP-D inhibited production of Th1 (TNF-α and IFN-γ) and Th17 (IL-17A) cytokines along with IL-6. Interestingly, levels of IL-2, Th2 (IL-4) and regulatory (IL-10 and TGF-ß) cytokines remained unaltered. Analysis of co-stimulatory CD28 and co-inhibitory CTLA4 receptors along with their ligands CD80 and CD86 revealed a selective up-regulation of CTLA4 in the lymphocyte subset. rhSP-D induced apoptosis in the activated but not in non-activated lymphocytes. Blockade of CTLA4 inhibited rhSP-D mediated apoptosis of activated lymphocytes, confirming involvement of CTLA4. We conclude that SP-D restores immune homeostasis. It regulates expression of immunomodulatory receptors and cytokines, which is followed by induction of apoptosis in activated lymphocytes. These findings suggest a critical role of SP-D in immune surveillance against activated immune cells.

Ovarian Hormones Regulate SP-D Expression in the Mouse Uterus During Estrous Cycle and Early Pregnancy.

PROBLEM: Differential expression of SP-D in the cycling human and mouse endometrium suggests its regulation by ovarian hormones. METHOD OF STUDY: SP-D expression in the mouse uterus was analyzed across the estrous cycle and during early pregnancy. Effect of exogenous ovarian hormones on the uterine expression of SP-D was analyzed. RESULTS: SP-D expression varied across the estrous cycle and peaked in the estrous phase. SP-D transcript levels increased by fourfold in the uteri of estrogen-treated mice while co-administration of estrogen and progesterone enhanced SP-D levels by ninefold. However, treatment with progesterone alone significantly downregulated SP-D expression. Diethylstilbestrol enhanced SP-D transcript levels in the uteri of immature mice by 10-fold. During pregnancy, SP-D levels declined rapidly from 0.5 dpc to 6.5 dpc. In silico analysis predicted the presence of two potential ERE and 1 PRE in the mouse SP-D gene promoter region. CONCLUSION: Estrogen positively regulates expression of SP-D in the mouse uterus. Progesterone, along with estrogen synergizes SP-D expression, however, when administered alone results in negative regulation.

Endometriotic mesenchymal stem cells exhibit a distinct immune phenotype.

Endometriosis is a significant debilitating gynecological problem affecting women of the reproductive age group and post-menopause. Recent reports suggest a role for endometriotic mesenchymal stem cells (ectopic MSCs) in the pathogenesis of endometriosis. To investigate the plausible mechanisms leading to the pathogenic behavior of ectopic MSCs, we compared the immunomodulatory properties of eutopic (healthy) and ectopic MSCs. We analyzed MSC phenotypes, differentiation potential, differential gene expression for an array of pattern recognition receptors (PRRs) and pro-inflammatory cytokine release along with markers of migration and angiogenesis among eutopic and ectopic MSCs. Further, alterations in immunosuppressive functions of eutopic and ectopic MSCs were examined by co-culturing them with mitogen-activated allogeneic PBMCs. Transcripts of PRRs such as all Toll-like receptors (TLR1-10), except TLR8, collectins (CL-L1, CL-P1 and CL-K1), NOD-1 and NOD-2 receptors and secreted pro-inflammatory cytokines like IL-6, IFN-γ, vascular endothelial growth factor (VEGF), epidermal growth factor and MCP-1 were significantly up-regulated in ectopic MSCs. The anti-inflammatory cytokine transforming growth factor-ß showed significant down-regulation, while IL-10 showed a significant increase in ectopic MSCs. Further, ectopic MSCs showed up-regulated expression for markers of migration and angiogenesis such as matrix metalloproteinase-2 (MMP-2), MMP-3 and MMP-9 and VEGF, respectively. We report here that proliferation of PBMCs was less inhibited upon co-culture with ectopic MSCs compared with eutopic MSCs. The findings suggest that ectopic MSCs with increased levels of TLRs, collectins, pro-inflammatory cytokines and markers of migration and angiogenesis exhibit a distinct immune phenotype compared to eutopic MSCs. This distinct phenotype may be responsible for the reduced immunosuppressive property of ectopic MSCs and may be associated with the pathogenesis of endometriosis.

Surfactant protein D inhibits HIV-1 infection of target cells via interference with gp120-CD4 interaction and modulates pro-inflammatory cytokine production.

Surfactant Protein SP-D, a member of the collectin family, is a pattern recognition protein, secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens. In this study, we confirm that native human SP-D and a recombinant fragment of human SP-D (rhSP-D) bind to gp120 of HIV-1 and significantly inhibit viral replication in vitro in a calcium and dose-dependent manner. We show, for the first time, that SP-D and rhSP-D act as potent inhibitors of HIV-1 entry in to target cells and block the interaction between CD4 and gp120 in a dose-dependent manner. The rhSP-D-mediated inhibition of viral replication was examined using three clinical isolates of HIV-1 and three target cells: Jurkat T cells, U937 monocytic cells and PBMCs. HIV-1 induced cytokine storm in the three target cells was significantly suppressed by rhSP-D. Phosphorylation of key kinases p38, Erk1/2 and AKT, which contribute to HIV-1 induced immune activation, was significantly reduced in vitro in the presence of rhSP-D. Notably, anti-HIV-1 activity of rhSP-D was retained in the presence of biological fluids such as cervico-vaginal lavage and seminal plasma. Our study illustrates the multi-faceted role of human SP-D against HIV-1 and potential of rhSP-D for immunotherapy to inhibit viral entry and immune activation in acute HIV infection.

Surfactant protein SP-D modulates activity of immune cells: proteomic profiling of its interaction with eosinophilic cells.

Surfactant protein D (SP-D), a C-type lectin, is known to protect against lung infection, allergy and inflammation. Its recombinant truncated form comprising homotrimeric neck and CRD region (rhSP-D) has been shown to bring down specific IgE levels, eosinophilia and restore Th2-Th1 homeostasis in murine models of lung hypersensitivity. SP-D knockout mice show intrinsic hypereosinophilia and airway hyper-responsiveness that can be alleviated by rhSP-D. The rhSP-D can bind activated eosinophils, inhibit chemotaxis and degranulation, and selectively induce oxidative burst and apoptosis in sensitized eosinophils. A global proteomics study of rhSP-D-treated eosinophilic cell line AML14.3D10 identified large-scale molecular changes associated with oxidative burst, cell stress and survival-related proteins potentially responsible for apoptosis induction. The data also suggested an involvement of RNA binding- and RNA splicing-related proteins. Thus, the proteomics approach yielded a catalog of differentially expressed proteins that may be protein signatures defining mechanisms of SP-D-mediated maintenance of homeostasis during allergy.