Despite tremendous advances in our understanding of HIV/AIDS since the first cases were reported 30 years ago, we are still a long way from understanding critical steps of HIV acquisition, pathogenesis and correlates of protection. Our new understanding of the importance of the mucosa as a target for HIV infection, as well as our recent observations showing that altered expression and responses of innate pattern recognition receptors are significantly associated with pathogenesis and resistance to HIV infection, indicate that correlates of immunity to HIV are more likely to be associated with mucosal and innate responses. Most of the heterosexual encounters do not result in productive HIV infection, suggesting that the female genital tract is protected against HIV by innate defence molecules, such as antiproteases, secreted mucosally. The present review highlights the role and significance of the serine protease inhibitors SLPI (secretory leucocyte protease inhibitor), trappin-2, elafin and ps20 (prostate stromal protein 20 kDa) in HIV susceptibility and infection. Interestingly, in contrast with SLPI, trappin-2 and elafin, ps20 has been shown to enhance HIV infectivity. Thus understanding the balance and interaction of these factors in mucosal fluids may significantly influence HIV infection.

HIV

Since the first case report of HIV/AIDS 30 years ago, tremendous advances have been made in changing the course of this pandemic, particularly with antiretroviral drug treatments. However, we are still a long way from having effective HIV preventive measures in place. Indeed, our understanding of critical steps in HIV/AIDS acquisition and pathogenesis remains poor, and climbing rates of HIV cases further confirm this notion. Having multiple cell targets of HIV infection as well as routes of viral transmission with their physiological/structural differences also adds complexity to designing effective preventive measures. The lack of clear immunological correlates of protection against HIV, partly attributable to the enormous global diversity of the virus, is another challenge in HIV prevention.

We are now aware that HIV transmission occurs primarily at mucosal sites; acute HIV infection is followed by explosive virus replication in T and Langerhan's cells as well as massive depletion of CD4+ T-cells in the mucosa, especially in the gut [1]. It is also clear now that HIV pathogenesis is driven by immune activation and high cell turnover [24]. Previously, the search for immune correlates in HIV was mainly focused on adaptive immune responses; however, in light of our new understanding of HIV pathogenesis, these correlates may be associated with mucosal and innate responses. Currently, the microbial translocation theory posits that immune activation in HIV is driven by a ‘leaky’ gut [5]. But our recent studies on CSWs (commercial sex workers) in Kenya indicated that untreated HIV infection is associated with significantly enhanced expression and responsiveness of innate PRRs (pattern-recognition receptors) that may be the penultimate driver of immune activation in HIV [6,7]. We also found that expression of selected PRRs was significantly altered in CMCs (cervical mononuclear cells) and CECs (cervical epithelial cells) of HIV-R (HIV-resistant) compared with HIV-S (HIV-susceptible) and HIV-P (HIV-positive) CSWs (X.D. Yao, R.T. Lester, R.W. Omage, B.M. Henrick, W. Jaoko, C. Wachihi, T.B. Ball, F.A. Plummer and K.L. Rosenthal, unpublished work). These observations further support our hypothesis that altered innate viral recognition and responsiveness at mucosal sites might be critical events, perpetuating immune activation and disease progression or resistance to HIV/AIDS.

It is still debatable whether ECs (epithelial cells) represent a direct target for HIV infection, regardless of their anatomical location. What is clear, however, is the significant role of genital ECs in early steps of HIV transmission. Indeed, the ability of genital ECs to directly recognize and respond to HIV may largely predetermine the outcome of the host–pathogen interaction [8,9]. Recent data indicate that women are disproportionately affected by this infection; however, most of the heterosexual encounters do not result in productive HIV infection [10]. This finding suggests that the FGT (female genital tract) is protected from HIV by endogenous defense molecules secreted mucosally and by multiple cells types, including ECs. In fact, we now know that CVLs (cervico-vaginal lavage samples) from healthy women show a pronounced activity against HIV and HSV (herpes simplex virus) that is attributed to the presence of cationic polypeptides [1113]. These protective innate factors belong to different groups, encompassing human defensins [14], cathelicidins [15] and antiproteases [1619]. Although members of these groups share limited homology in structure, they are closely related in their broad antimicrobial and immunomodulatory properties.

General characteristics of antiproteases SLPI (secretory leucocyte protease inhibitor), Tr (trappin-2) and E (elafin)

The group of neutrophil serine protease inhibitors includes nearly 20 members, but the nature and properties of SLPI, Tr and E are characterized the most. All three, SLPI, Tr and E, belong to the WAP (whey acidic protein) family of polypeptides that share a domain with eight characteristic cysteine residues that form four disulfide bonds, thus the name ‘four-disulfide core’ domain [20]. The FDC (four-disulfide core) domain is also called a WAP domain, as it is similar to a whey acidic protein, initially identified from rodent milk [21]. Hence FDC-containing proteins from a WAP group are often referred to as WFDC (WAP FDC) proteins. However, note that it is not only WAP proteins that have an FDC domain [20,22], hence calling all proteins with an FDC domain WFDC proteins is inaccurate.

SLPI is a non-glycosylated cationic 11.7 kDa protein of 107 amino acids and two WAP domains, as reviewed in [23]. In the early 1970s, SLPI was originally isolated from bronchial secretions [24] and was later found to have antiprotease, antimicrobial and anti-inflammatory properties and to be present in different cell types and locations, including FGT [22,23]. SLPI shows inhibitory activity against neutrophil serine proteases cathepsin G and neutrophil elastase that appears to be linked to the second WAP domain. Tr is another unglycosylated protein of 95 amino acids (9.9 kDa) that has E, as a C-terminal WAP domain of 57 amino acids (5.9 kDa) [25] and an N-terminal cementoin domain of 38 amino acids. Tr and E exhibit inhibitory properties against neutrophil serine proteases elastase and proteinase 3 [23]. E is generated by proteolytic cleavage of the N-terminus cementoin domain of Tr by mast cell tryptase [26], and appears to share 40% homology with SLPI [23].

Serine antiproteases SLPI, Tr and E are detectable in both serum and mucosal secretions [27], and are present in many cell types and tissues [28], including genital ECs, where they are secreted in response to pro-inflammatory stimuli [16]. Interestingly, we recently confirmed and extended a previous observation [16] by showing that human endometrial cells HEC-1A secreted both Tr and E in response to a synthetic mimic of viral dsRNA (double-stranded RNA), poly(I:C) (polyinosine-polycytidylic acid) (A.G. Drannik, K. Nag, X.-D. Yao, B.M. Henrick, J.-M. Sallenave and K.L. Rosenthal, unpublished work). Since dsRNA is considered to be a by-product of many, if not all, virus infections [29], our finding may indicate that in the presence of viral antigens, both Tr and E are present in CVL and act against viruses in the FGT.

Similar to SLPI, Tr and E primarily function to protect tissues from excessive proteolysis due to neutrophil activation [30,31] and multiple pathogens via direct or indirect mechanisms [16,3235]. Anti-inflammatory features of these serine antiproteases were also extensively characterized, but primarily in response to bacterial antigens. Specifically, SLPI, Tr and E were shown to reduce the expression of pro-inflammatory factors as a result of their inhibitory effect on NF-κB (nuclear factor κB) and AP-1 (activating protein 1) by altering IκB (inhibitor of NF-κB) activation [36] and proteasomal degradation [37] respectively. Worth mentioning is that we also showed recently that both Tr and E were capable of reducing pro-inflammatory factors IL-8 (interleukin-8), IL-6 and TNFα (tumour necrosis factor α) as well as antiviral IFNβ (interferon β) in response to poly(I:C) (Figure 1). Our results also demonstrated that Tr and E significantly inhibited VSV (vesicular stomatitis virus)-GFP (green fluorescent protein) replication, directly or indirectly, and enhanced poly(I:C)-induced antiviral protection (A.G. Drannik, K. Nag, X.-D. Yao, B.M. Henrick, J.-M. Sallenave and K.L. Rosenthal, unpublished work). Interestingly, enhanced antiviral protection was not associated with higher induction of IFNβ in the present study. We showed further that reduced inflammatory factors in the presence of Tr and E were associated with lower expression of innate viral sensors RIG-I (retinoic acid-inducible gene I) and MDA-5 (methylene dianiline 5; Figure 1). We believe this was the first evidence of Tr and E affecting host innate recognition and modulation of antiviral and inflammatory responses in genital ECs that is significant for health and disease of the female reproductive tract (A.G. Drannik, K. Nag, X.-D. Yao, B.M. Henrick, J.-M. Sallenave and K.L. Rosenthal, unpublished work). More recently, however, dual immunomodulatory properties of Tr and E were also reported; that is, depending on the environment, Tr and E can either dampen inflammation [37,38] or promote immunostimulatory events and prime the immune system [39,40], suggesting that we still do not understand all the conditions and factors regulating Tr and E functions.

Tr and E inhibit the expression of innate viral sensors RIG-I and MDA-5 as well as pro-inflammatory and anti-viral mediators in genital epithelial cells in response to a mimic of viral dsRNA, poly(I:C).

Anti-HIV activity of SLPI, Tr and E

Interest in SLPI, Tr and E continues to grow as more evidence is accumulated on their anti-HIV properties [16,19,41]. Initially, it was discovered that low HIV transmission through saliva and CVL was correlated with high levels of SLPI in the mucosal fluids. Subsequently, the potent anti-HIV activity of SLPI was shown to be independent of its antiprotease activity and mediated through its direct effect on cells, but not HIV [41]. Specifically, two potential modes of anti-HIV action of SLPI have been described to date: (i) SLPI interferes with HIV fusion with the T-cell plasma membrane through binding to scramblase 1, a membrane protein that interacts with CD4 and controls the movement of the phospholipid bilayer of the plasma membrane [42]; (ii) SLPI blocks viral entry/fusion with a myeloid cell as a result of binding to annexin II, a macrophage receptor for a phosphatidylserine moiety that HIV carries on its outer coat on exiting from an infected cell [34]. Interestingly, SLPI appears to be protective against HIV only in models using monocytes/macrophages and T-cells, but not genital ECs, despite the fact that SLPI is also secreted by genital ECs [43]. The lack of protection on ECs could suggest that anti-HIV activity of SLPI might be limited or predetermined by the cell or a receptor type, on which HIV is acting. In other words, SLPI might have differential ability to block HIV receptors, depending on their types, cell distribution and SLPI affinity to them.

Considering the previously demonstrated resemblance between SLPI, Tr and E in structure and function, one would anticipate that anti-HIV features of the antiproteases would also be similar. Indeed, E was recently identified as a biomarker of HIV resistance in CSWs in Kenya [16,19,32,41]. Iqbal et al. have demonstrated using SELDI-TOF (surface-enhanced laser-desorption ionization-time-of-flight) MS that expression of a 6 kDa protein E was significantly higher in CVLs of HIV-R CSWs, compared with HIV-S controls [19]. Although no specific mechanism of action was proposed, in their patent the investigators showed that recombinant E prevented the infection of T-cells in vitro [44]. Interestingly, however, when HIV-S CSWs were followed prospectively, the authors reported that elevated levels of both Tr and E were associated with HIV protection. This finding indicates that both proteins might be required for optimal protection against HIV [19], which warrants additional investigation in the future. Recently, another group has also shown a protective effect of E against HIV and suggested that it was the result of a direct effect of E on HIV [16].

In agreement with the above data, we also showed recently that rTr/rE (recombinant Tr and E) each independently inhibited HIV attachment and transcytosis across human genital ECs in vitro. Treatment of HIV or cells with rTr/rE significantly decreased virus attachment to ECs, suggesting direct and indirect mechanisms of anti-HIV activity of Tr and E (A.G. Drannik, X.-D. Yao, B.M. Henrick, K. Nag, S. Jain and K.L. Rosenthal, unpublished work). Furthermore, we also confirmed significantly higher expression of Tr and E in CVLs of HIV-R, and this was associated with reduced mRNA of TLR-2, TLR-4 and RIG-I in ECs from HIV-R compared with HIV-S, as determined by quantitative reverse transcription–PCR. Collectively, these findings highlight important and multifaceted role(s) of Tr and E in innate viral recognition and ultimately protection against HIV in FGT.

ps20: an HIV-enhancing factor

Recently, another serine antiprotease was implicated in HIV [45,46]. Similar to SLPI, Tr and E, ps20 (prostate stromal protein 20 kDa) belongs to the WAP family of proteins that share FDC domain [46] and participates in wound repair and cell migration. However, in contrast with SLPI, Tr and E, ps20 does not possess antiprotease activity and has also been identified as a pro- or enhancing HIV factor, or marker of HIV permissiveness of CD4+ memory T-cells. Human ps20 was initially purified from the prostate gland stroma as a growth inhibitor and a correlate of stromal-epithelial cell differentiation and interaction [47,48]. Primary biological functions of ps20 relate to cancer inhibition, angiogenesis, regulation of extracellular matrix, cell migration and wound repair [4749]. Interestingly, HIV appears to make ps20 a Trojan horse. Specifically, HIV utilizes the ability of ps20 to affect cell–cell adhesion and migration through the activation of the LFA-1 (lymphocyte function-associated antigen 1)/CD54 integrin pathway by up-regulating expression of CD54 [ICAM-1 (intercellular adhesion molecule 1)], thus making CD4+ memory T-cells more susceptible to infection. This integrin pathway has been shown to be important for normal cell conjugation. Hence, its activation promotes cell–cell adhesion and more efficient intercellular HIV transfer and entry into CD4+ T-cells [46].

A WAP protein axis in HIV?

From the evidence presented in the present review, it is clear that, despite having common familial roots, SLPI, Tr, E and ps20 exhibit differential functions in the context of HIV (Table 1). With protective anti-HIV effects of SLPI, Tr and E on the one hand and pro-HIV action of ps20 on the other, one might wonder whether HIV susceptibility and infection could be the result of imbalanced ratios of WAP proteins at routes of viral entry. Could there be an axis, or a yin yang, in WAP proteins and susceptibility to HIV infection (Figure 2)? The answer to this question is unclear at the moment, since we still have limited understanding of how these serine antiproteases interact and whether their targets and mechanisms of actions overlap. In other words, we do not really know whether SLPI, Tr and E can regulate the expression of adhesion molecules, such as ICAM-1 and LFA-1, and whether SLPI, Tr and E can negate the enhancing effect of ps20 on HIV by simply changing the expression of the integrins. Clearly, the ubiquitous expression of SLPI, Tr, E and ps20, their contribution to host homoeostasis and the emerging evidence of their role in HIV infection underscore an urgent need for more research in future on the role of these molecules in HIV. It might be important to know the patterns and ratios of expression between SLPI, Tr, E and ps20 in mucosal fluids or the same HIV target cells, and whether there is a relationship between altered, or imbalanced, homoeostatic expression of SLPI, Tr, E and ps20 and HIV susceptibility. In addition, it might be of significance to know whether ps20 is secreted mucosally and whether this enhancing effect of ps20 on HIV can be relevant to ECs, given the primarily mucosal/sexual nature of HIV transmission.

Table 1
Functions of SLPI, Tr, E and ps20 in addition to their antiprotease activity
Action Reference(s) 
SLPI  
 Antibacterial, antifungal, antiviral (HIV), anti-inflammatory, tissue repair [2224,32,34,36,49
Tr and E  
 Antibacterial, antifungal, Anti-inflammatory, tissue repair, priming innate system [22,23,33,3542,44,49
 Antiviral (HIV) [16,19
 Antiviral (VSV, HIV), inhibition of viral innate sensors (A.G. Drannik, X.-D. Yao, B.M. Henrick, K. Nag, S. Jain and K.L. Rosenthal, unpublished work) 
ps20  
 Growth inhibition, pro-angiogenic, pro-viral (HIV), cell recruitment, migration, adhesion, cancer suppressor, regulation of extracellular matrix [22,4549
Action Reference(s) 
SLPI  
 Antibacterial, antifungal, antiviral (HIV), anti-inflammatory, tissue repair [2224,32,34,36,49
Tr and E  
 Antibacterial, antifungal, Anti-inflammatory, tissue repair, priming innate system [22,23,33,3542,44,49
 Antiviral (HIV) [16,19
 Antiviral (VSV, HIV), inhibition of viral innate sensors (A.G. Drannik, X.-D. Yao, B.M. Henrick, K. Nag, S. Jain and K.L. Rosenthal, unpublished work) 
ps20  
 Growth inhibition, pro-angiogenic, pro-viral (HIV), cell recruitment, migration, adhesion, cancer suppressor, regulation of extracellular matrix [22,4549

The HIV-modulating effect of WFDC family proteins Tr, E, SLPI and ps20

Figure 2
The HIV-modulating effect of WFDC family proteins Tr, E, SLPI and ps20

(1) Tr and E inhibit HIV transcytosis through a monolayer of genital ECs by reducing attachment of virus to ECs due to direct anti-HIV and indirect effects of Tr and E; (2) E inhibits HIV infection of CD4+ T-cells by potentially binding to HIV binding sites on T-cells; (3) SLPI inhibits HIV infection of macrophages by blocking binding between PS (phosphatidylserine) and annexin II; (4) SLPI inhibits HIV infection of CD4+ T-cells by preventing binding between HIV and CD4 through the formation of SLPI–scramblase complexes; (5) ps20 enhances HIV infection of CD4+ T-cells by promoting HIV entry via fusion as a result of increased expression of cell-surface CD54 and thus increases cell-free and cell–cell spread of HIV.

Figure 2
The HIV-modulating effect of WFDC family proteins Tr, E, SLPI and ps20

(1) Tr and E inhibit HIV transcytosis through a monolayer of genital ECs by reducing attachment of virus to ECs due to direct anti-HIV and indirect effects of Tr and E; (2) E inhibits HIV infection of CD4+ T-cells by potentially binding to HIV binding sites on T-cells; (3) SLPI inhibits HIV infection of macrophages by blocking binding between PS (phosphatidylserine) and annexin II; (4) SLPI inhibits HIV infection of CD4+ T-cells by preventing binding between HIV and CD4 through the formation of SLPI–scramblase complexes; (5) ps20 enhances HIV infection of CD4+ T-cells by promoting HIV entry via fusion as a result of increased expression of cell-surface CD54 and thus increases cell-free and cell–cell spread of HIV.

Concluding remarks and future directions

The present review highlights the significance of innate mucosal factors SLPI, Tr, E and ps20 in mediating HIV susceptibility and infection. Building on our current but limited understanding of the inhibitory effects of Tr, E and SLPI versus the enhancing effects of ps20 on HIV, we propose a potential model of how these innate factors may influence HIV transmission in the FGT through their modulation of viral recognition and mounting of antiviral immune-inflammatory responses (Figure 2).

Although recent efforts in HIV vaccine trials provide a glimmer of hope that an effective prophylactic vaccine can be developed, it may take a decade before it becomes widely available [50]. Therefore efforts to continuously advance our understanding of the role of innate mucosal factors or biomarkers of resistance to HIV, such as the WAP proteins, will inform mucosal HIV vaccine development.

Structure and Function of Whey Acidic Protein (WAP) Four-Disulfide Core (WFDC) Proteins: An Independent Meeting held at Robinson College, Cambridge, U.K., 12–14 April 2011. Organized and Edited by Colin Bingle (Sheffield, U.K.), Judith Hall (Newcastle, U.K.), Cliff Taggart (Queen's University Belfast, U.K.) and Annapurna Vyakarnam (King's College London, U.K.).

Abbreviations

     
  • CSW

    commercial sex worker

  •  
  • CVL

    cervico-vaginal lavage sample

  •  
  • dsRNA

    double-stranded RNA

  •  
  • E

    elafin

  •  
  • EC

    epithelial cell

  •  
  • FDC

    four-disulfide core

  •  
  • FGT

    female genital tract

  •  
  • HIV-R

    HIV-resistant

  •  
  • HIV-S

    HIV-susceptible

  •  
  • ICAM-1

    intercellular adhesion molecule 1

  •  
  • IFNβ

    interferon β

  •  
  • IL

    interleukin

  •  
  • LFA-1

    lymphocyte function-associated antigen 1

  •  
  • MDA-5

    methylene dianiline 5

  •  
  • poly(I:C)

    polyinosine-polycytidylic acid

  •  
  • PRR

    pattern-recognition receptor

  •  
  • ps20

    prostate stromal protein 20 kDa

  •  
  • RIG-I

    retinoic acid-inducible gene I

  •  
  • rTr/rE

    recombinent trappin-2 and elafin

  •  
  • SLPI

    secretory leucocyte protease inhibitor

  •  
  • TNFα

    tumour necrosis factor α

  •  
  • Tr

    trappin-2

  •  
  • WAP

    whey acidic protein

  •  
  • WFDC

    WAP FDC

We thank K. Nag, X.-D. Yao, S. Jain (all at McMaster University) and J.-M. Sallenave (Institut Pasteur, Paris, France) for research assistance and the expert technical support of J. Newton and A. Patrick (both at McMaster University).

Funding

This work was supported by a grant as part of the Comprehansive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC), a key component of the Collaboration for AIDS Vaccine Development (CAVD) funded by the Bill and Melinda Gates Foundation. A.G.D. was supported by a studentship from the Otario HIV Treatment Network (OHTN) and K.L.R. was supported by a Career Scientist Award from the OHTN.

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