Adapted from Journal of Internal Medicine 2009;265(1):18-28
Part 1: Langerin and DC-SIGN: universal pathogen receptors?
In this thesis the function of LCs and DC-SIGN+ DCs in innate defense against
pathogens was investigated. Since LCs and DCs express distinct C-type lectins
Langerin and DC-SIGN, respectively, the interaction of different pathogens with these
C-type lectins was explored and resulted in the identifi cation of novel ligands and
pathogens. Thus far, HIV-1, Candida albicans and Mycobacterium leprae have been
identifi ed as ligands for Langerin (1-4). In this thesis it was shown that Langerin as
well as DC-SIGN interact with HSV-1 and -2 (chapter 4 and 8). Furthermore, fungal
species Malassezia furfur, Saccharomyces cerevisiae and a plethora of Candida
species were identifi ed as ligands for Langerin (chapter 2). These data suggest
that Langerin, similar to DC-SIGN, is a pattern recognition receptor with a broad
specifi city for pathogens and this raises the question whether the immunological and
virological outcomes are also similar.
1.1 Pathogen recognition
Langerin and DC-SIGN have overlapping carbohydrate specifi cities; they both
interact with mannose and fucose structures (5;6). Many viruses and other micro-
organisms express (host-cell derived) mannose structures on their surface, which
facilitate recognition by Langerin and DC-SIGN (7-9). Therefore, based on the
carbohydrate structure of ligands, one can make a prediction about the binding
capacity to Langerin and DC-SIGN.
Langerin has high affi nity for mannose, fucose and GlcNAc structures (6).
However, Langerin does not interact with LeX and LeA, while it has strong affi nity for
LeY and LeB (chapter 5). LeY and LeB contain a terminal fucose structure, which is
absent in LeX and LeA. These data suggest that Langerin is unable to bind internal
fucose structures, but does interact with terminal fucose structures. In contrary, DC-
SIGN interacts with all above mentioned Lewis structures (5). This is an apparent
difference between the binding capacities of Langerin and DC-SIGN, and shows that
Langerin has a distinct recognition pattern for fucose structures.
Fucose structures are often present on helminths, and may vary during the life-
stage. As seen in table 1, several helminths interact with Langerin and/or DC-SIGN.
However, distinct patterns are present: whereas Langerin recognizes Haemoncus
contortus larvae, these are not recognized by DC-SIGN. On the other hand, DC-
SIGN strongly interacts with cercariae from Schistosoma mansoni, while Langerin
fails to interact with these cercariae. S. mansoni cercariae are the fi nal larvae stage,
which penetrates the skin of the human host. Upon entry, cercariae rapidly transform
into a schistosomule, which is accompanied by profound changes in the antigenic
structures and the schistosomule migrates throughout the tissues to the lung and
liver (10). The fi nding that cercariae do not inte ract with Langerin suggests that this is
a possible mechanism for the parasite to escape recognition by LCs. Together these
data suggest that structures present on helminths and other pathogens determine
the recognition by Langerin and DC-SIGN, which, dependent on the subsequent
immune reaction, may provide an immune-evasive strategy.
Langerin interacts with the cell-wall of fungal pathogens, which contain mannose
as well as β-glucan structures (chapter 2). Mannose structures present on fungal
cell-walls are also ligands for DC-SIGN (5;6). Notably, Langerin interacts with both
curdlan and laminarin, structures containing mainly β1-3 glucans. Thus far, dectin-1,
present on DCs and expressed at low levels by LCs, has been the only C-type
lectin identifi ed to interact with β-glucans (11). Recently, α-glucans isolated from
M. tuberculosis have been identifi ed as a ligand for DC-SIGN (12). It remains to be
determined whether Langerin interacts with α-glucans. In addition to the identifi cation
of Langerin as a receptor for β-glucans and fungi, these data show that Langerin
acts as the main fungal receptor on LCs. Thus, these studies suggest that Langerin
has a broad pathogen recognition pattern.
As listed in table 1, several pathogens fail to interact with Langerin and/or DC-
SIGN. Together these data demonstrate the importance of carbohydrate structures
that are present on pathogens in the recognition by Langerin and DC-SIGN. Extensive
trimming of glycans present on a pathogen may provide an immune-evasive strategy
in the interaction with Langerin and/or DC-SIGN. For instance, HBV is unable to
interact with DC-SIGN: however, viral particles produced in the presence of an
inhibitor that prevents trimming of complex mannose structures are able to interact
with DC-SIGN (chapter 9).
Although Langerin and DC-SIGN recognize a large variety of pathogens and show
overlapping recognition patterns, they have unique specifi cities that helps to diversify
immune recognition. This is further supported by the differences in expression and
1.2 Host-pathogen interaction
Host-pathogen interaction can have a wide variety of outcomes. DCs are essential
in shaping innate and adaptive immune responses to pathogens. In this section the
interaction of pathogens with LCs and DCs and the contribution of C-type lectins DC-
SIGN and Langerin are discussed (Figure 1).
1.2.1 DC-SIGN+ DCs
C-type lectins on DCs play an important role in pathogen recognition for
antigen-presentation. Antigens are taken up by C-type lectins such as DC-SIGN
and targeted to the lysosome for (cross-)presentation onto MHC-I or -II (21). This
requires an effi cient maturation of DCs. However, several pathogens have evolved
to subvert the antigen processing capacity and inhibit DC maturation. For example,
HSV-1 actively down-regulates co-stimulatory molecules and MHC-I, which results
in suppression of DC maturation and impaired antigen-presentation, while Measles
Virus (MV) prevents CD8+ T cell proliferation (22).
Another mechanism by which pathogens evade DC-mediated immunity is by
interfering with antigen processing routing to not only prevent antigen presentation,
but to also enhance pathogen survival and dissemination. HCV binding to DC-
SIGN leads to targeting of HCV to non-lysosomal compartments, while antibodies
and carbohydrates are normally directed through early endosomes to lysosomes
(18;21). Binding to DC-SIGN also results in re-direction of the internalization route to
Table 1: Langerin and DC-SIGN binding
terminal-fucose, GlcNAc (6)
internal/ terminal fucose (5)
LeA, LeB, LeX, LeY (5)
Soluble egg antigen
M. tuberculosis (ManLam)
HIV-1 gp120 R5/ X4
Salp15 (Ixodes scapularis) Yes Yes (20)
nd: not determined
non-lysosomal compartments for protection and transmission of pathogens to target
cells (23). This is benefi cial to the pathogen since it would prevent effi cient antigen
presentation and subsequent immune stimulation, and in addition it could provide a
mechanism by which pathogen transmission throughout the body occurs. It has been
demonstrated that pathogens such as HIV-1 and M. tuberculosis survive within DCs
(15;24;25) and that HIV-1 is subsequently transmitted to target cells, a mechanism
known as trans-infection (15;24;25).Viruses exploit DC-SIGN binding to facilitate
attachment and thereby enhance infection of the host. Several pathogens including
HIV-1, MV, and HSV-1 exploit DC-SIGN binding for enhanced infection of DCs and
transmission to target cells (15;16;19). Along these lines, the lack of binding to DC-
SIGN can also provide a mechanism to escape from DC immune surveillance, such
as demonstrated for HBV.
Interaction of pathogens with DCs often involves simultaneous activation of
TLRs and C-type lectins and co-triggering of TLR and C-type lectin tailors immune
responses (26). Synergistic C-type lectin and TLR activation results in altered
cytokine production, which in turn can modulate T cell proliferation and T helper
(Th) cell differentiation. Likewise, DC-SIGN and TLR-4-mediated tailoring of immune
responses has been thoroughly investigated (27); stimulation of DC-SIGN alone fails
to induce a cytokine response, while upon activation of TLR-4, DCs produce cytokines
such as IL-10, IL-12 and IL-6. However, engagement of mannose structures with
DC-SIGN together with TLR ligation enhances the production of IL-10 as well as
pro-infl ammatory cytokines IL-12 and IL-6 in DCs (28). Whereas TLR signaling is
responsible for the recruitment of NF-ĸB to the nucleus, DC-SIGN triggering results
in phosphorylation and subsequent acetylation of NF-ĸB subunit p65 via a signaling
pathway including a signalosome containing LSP-1 and Raf-1. Together this results
in prolonged and enhanced transcription of IL10, IL12p35 and IL6. NF-ĸB modulation
Figure 1. Immune evasion and modulation by pathogens. Pathogens evade and modulate host
immune responses through several mechanisms. (A) Pathogens interfere with internalization, maturation
and antigen presentation through receptors such as DC-SIGN. Both TLR and C-type lectin receptors
interact with pathogens, which induces signaling that tailors the immune response. (B) Pathogen binding to
DC-SIGN enhances attachment and subsequent DC infection (cis-infection). De novo produced particles
are released and transmitted to target cells (1). Lack of interaction with DCs can lead to evasion from
immune recognition (2). (C) Pathogen binding to DC-SIGN enhances infection of target cells independent
of DC infection (trans-infection). This is mediated via cell-surface bound (1) or internalized (2) particles.
has been demonstrated for TLR-3, TLR-4 and TLR-5 stimulation in combination
with DC-SIGN (27). In contrast, engagement of fucose structures together with TLR
ligation results in enhanced IL-10, but decreased IL-6 and IL-12 production (28).
DC-SIGN triggering by fucose structures results in dissociation of the signalosome
containing Raf-1 from the cytoplasmic tail of DC-SIGN. The mechanism responsible
for the fucose-specifi c cytokine profi le remains unclear, although it is known that
this process requires LSP-1, but not Raf-1, in the signalosome. Thus, DC-SIGN
tailors TLR signaling by responding differently to mannose and fucose carbohydrate
Helicobacter pylori is a human gastric pathogen that exists in two different phase
variants, LeY+ and LeY- (29). Phase switching can be used by the pathogen as a
mechanism to subvert the immune response by DCs. Indeed, LeY+ bacteria increase
anti-infl ammatory cytokine IL-10 and suppress pro-infl ammatory cytokines IL-12 and
IL-6 production compared to LeY- bacteria (28). This could account for the observed
suppression of Th1 response during H. pylori infection (29). DC-SIGN signaling in
response to LeY+ variants can thus redirect the immune response towards a more
balanced Th1/Th2 response, which is benefi cial for the host compared to a dominant
Th1 response, which has been associated with increased gastric infl ammation
(29;30). Thus, phase switching to a LeY- variant would be more detrimental for the
host and advantageous for the parasite compared to LeY+ variants.
In addition to mannose and fucose induced DC-SIGN signaling, another
mechanism is observed for Salp15. Salp15 is a salivary protein isolated from Ixodes
scapularis (deer tick), the primary vector for Borrelia burgdorferi transmission. In
chapter 10, Salp15 and whole tick saliva inhibited T cell proliferation upon TLR
stimulation in DCs. Salp15 interaction with DC-SIGN on DCs synergistically with
TLR-2 or TLR-4 triggering leads to suppression of pro-infl ammatory cytokines IL-12,
IL-6 and TNF-α. This suppressed cytokine production is dependent on intracellular
signaling molecules Raf-1 and MEK. The decreased IL-6 and TNF-α production is
caused by enhanced mRNA degradation, while IL-12 production is hampered due
to impaired nucleosome remodelling at the IL12-p35 promoter. This mechanism
appears to be distinct from that of mannose or fucose structures and is not dependent
on NF-kB phosphorylation or acetylation. However, the exact mechanism remains
to be elucidated. Salp15 has been shown to bind to CD4, thereby inhibiting T cell
receptor ligation-induced signals, resulting in reduced IL-2 production and impaired
CD4+ T cell activation and proliferation (31;32). Salp15 may alter the localization of
intracellular signaling molecules involved in DC-SIGN signaling. In addition, other
receptors, such as CD4 might affect the localization of Raf-1 or other downstream
effector molecules to DC-SIGN. Together, the immunosuppressed environment
created by tick saliva including Salp15 might facilitate B. burgdorferi‘s escape from
DCs and decreases activation of T cells. Subsequently, the immunosuppressed
environment is advantageous for the dissemination of B. burgdorferi through the body.
Thus, immune responses mediated by DC-SIGN are tailored through regulation of
signaling pathways after triggering of different receptors or by ligands with different
1.2.2 Langerin+ LCs
As described previously, the interactions of pathogens with DC-SIGN have been
thoroughly investigated. However, less is known about the interaction of Langerin
and LCs with micro-organisms. In this thesis several pathogens have been identifi ed
as ligands for Langerin, including HSV-1 and -2, mycobacteria, helminths, and fungi,
such as Candida species, Saccharomyces cerevisiae and Malassezia furfur (Table
1). In this section the interaction of LCs with different pathogens is discussed.
Opposite to DC-SIGN, the interaction of HIV-1 with Langerin does not result in
enhanced infection, but rather results in degradation of HIV-1 by targeting HIV-1 to
Birbeck granules (1). Unpublished observations suggest that a similar mechanism
plays a role in interaction of Langerin with MV. MV binding to Langerin decreases
infection of LCs (L. de Witte, unpublished observations). It is likely that HSV-1 and -2
interactions with Langerin also results in targeting to the Birbeck granules. Electron
microscopy analysis could provide better insight. An important difference between
HSV and HIV-1 is that HSV is highly infectious for LCs, surrounding keratinocytes
and epithelial cells. In addition, LCs express other surface molecules with high
affi nity for HSV, such as heparan sulfates, which facilitate attachment of HSV to LCs
(33;34). Therefore, it is plausible that under physiological circumstances Langerin on
LCs might be less effi ciently to prevent HSV infection of LCs.
HIV-1 and MV investigations suggest that Langerin binding results in targeting
of these viruses to Birbeck granules for degradation. It remains to be determined
whether this holds true for all Langerin-binding viruses and whether other micro-
organisms can also be targeted to the Birbeck granules. Electron microscopic
analysis of the interaction of C. albicans with Langerin demonstrates uptake of C.
albicans by LCs via Langerin, although no internalization in Birbeck granules is
observed (chapter 2). Indeed, the large size of the particles raises questions about
the possibility of uptake of C. albicans into Birbeck granules.
LCs are also thought to play a role in antigen presentation. The classical paradigm
has been that LCs have similar antigen presenting capacities as their DC-SIGN+ DC
counterparts. However, little evidence is available to date to support this. There are
confl icting data about the role of murine LCs in inducing immune responses and
careful interpretation is needed. The results of different studies are dependent on the
stimuli and model used, especially since the recent identifi cation of murine Langerin+
CD103+ dermal DCs that seem to be involved in certain processes previously
attributed to LCs (35).
Current data do not support a prominent role for LCs in the induction of immune
responses: LCs appear to be dispensable for immune activation such as contact
hypersensitivity (36-38), graft-versus-host disease (39) and viral immunity (40;41).
Together, this has lead to the hypothesis that LCs may have immune-regulatory
properties that serve to constrain and contain adaptive immune responses in the
skin. However, the majority of these studies have been performed in mice and under
different conditions that might affect the outcome. Indeed, LC function in these mouse
models might be dependent on the nature of the antigen, the type of pathogen, and
the immunization route (42).
Murine studies have shown that LCs present antigens on MHC-I and MHC-II (43),
however, LCs fail to cross-present viral antigens. In a murine HSV model, LCs do
not present HSV antigens to CD8+ T cells in lymph nodes. Instead it is thought that
LCs transfer their antigens or apoptotic LC fragments to another DC subset (40).
Whether this antigen transfer takes place in the epidermis, dermis or in the lymph
nodes remains unclear (40;44). In a human MV-specifi c antigen presentation model,
it is observed that LCs effi ciently present antigens to CD4+ T cells and that antigen
presentation onto MHC-I occurs after viral infection of LCs. However, LCs fail to
cross-present antigens to CD8+ T cells (L. de Witte, unpublished observation). In
contrast, LCs are able to transfer antigens to DC-SIGN+ DCs, which subsequently
cross-present antigens to CD8+ T cells, which is independent of viral infection
(de Jong, unpublished observation). These data support the hypothesis that LCs
transfer viral antigens to DCs for further processing and presentation. Thus, LCs
effi ciently present viral antigens onto MHC-II and onto MHC-I upon viral infection. To
overcome the lack of ability to cross-present antigens, LCs transfer viral antigens to
DCs for cross-presentation. It remains to be determined whether this is specifi c for
Very little is known about the role of Langerin in antigen presentation. Targeting
of Langerin with monoclonal antibodies results in delivery to non-lysosomal
compartments but not to MHC-II molecules (45). In addition, Langerin-mediated
antigen uptake results in delivery to Birbeck granules and may be involved in CD1a
dependent antigen presentation (4). Nevertheless, in mice it has been demonstrated
that LCs present OVA-labelled anti-Langerin antibodies both to CD4+ and CD8+ T
cells (43). It remains to be determined whether pathogens captured by Langerin
are similarly processed and presented to both CD4+ and CD8+ T cells, and whether
this occurs in human LCs. Preliminary data suggest at least a partial contribution
of Langerin in MV uptake for MHC-II presentation by human LCs (L. de Witte,
The characteristics of LCs to deliver antibodies to Birbeck granules, which are
thought to be involved in the endosomal recycling pathway, is very distinct from
DC-SIGN and not completely understood. Langerin has a characteristic proline
rich sequence in its cytoplasmic tail (46), while DC-SIGN bears a cytoplasmic tail
containing internalization motifs with a di-leucine motif and a tri-acidic clusters
(23). The differences in cytoplasmic tails can lead to recruitment of distinct adaptor
proteins for each lectin, which are involved in internalization, intracellular sorting and
even cytokine responses. It is currently unknown whether Langerin signaling affects
adaptive immune responses and how the internalization pathway by Langerin is
regulated at the molecular level.
LCs have a restricted localization within the human body: they line the epidermis
and mucosal tissues, which are constantly exposed to the environment. The
localization suggests that LCs play a regulatory role while DCs in the sub-epithelium
and dermis, located below this delicate layer of LCs, play an important role in defense
against pathogens. Based on the strategic location and compartmentalization of LCs
and DCs it is tempting to speculate that high responsiveness of DCs to pathogens is
necessary when the primary defense barrier is damaged and a pathogen has invaded
the body. On the other hand, LCs are constantly exposed to micro-organisms in our
environment and should therefore respond selectively. Several observations have
been reported to support this hypothesis.
In general, LCs show a delayed migration from the epidermis to the lymph nodes
compared to dermal DCs (38). These data have led to the hypothesis that LCs do
not play a pivotal role in early antigen presentation events during infection. However,
the unpublished observations demonstrating that LCs are capable of transferring
viral antigens for cross-presentation to DCs, suggest that LCs do play a role in innate
defense against viral pathogens. Therefore, antigen transfer may take place in the
dermis. Further research is needed to clarify the functions of these cells.
Differences in C-type lectin and TLR expression contribute to the divergent
function of LCs and DCs. Langerin and DC-SIGN have distinct functions in HIV-
1 infection: DC-SIGN enhances infection and transmission, whereas Langerin
captures HIV-1 for degradation. Under non-infl ammatory conditions LCs form a fi rst
line of defense against HIV-1 infection. In contrast, HIV-1 subverts highly migratory
DCs for transmission throughout the host. The interaction of LCs and DCs with HIV-1
is discussed in more detail in part 2 of this discussion. It remains to be determined
whether this also holds true for other viruses and other classes of micro-organisms.
The repertoire of TLRs expressed by LCs is distinct from that expressed by
DCs. Whereas DCs express TLRs that recognize bacteria, fungi and viruses, LCs
lack expression of TLR-4, which recognizes gram-negative bacteria (47;48). In
general, bacterial vaginosis is mainly caused by gram-negative bacteria. In addition,
the responsiveness of LCs to bacterial TLR ligands is weak in terms of cytokine
production and in general LCs produce low amounts of cytokines. LCs produce
a repertoire of cytokines including IL-6, IL-8 and TNF-α but not IL-12 and type I
interferons (47). Nevertheless, TLR-2 triggering of LCs induces IL-10 production,
which suggests a specifi c response to commensal gram-positive bacteria (47). LCs
do not respond to lactobacilli present in the healthy vaginal fl ora. This gram-positive
bacterium is abundantly present, but does not usually cause an overt infl ammatory
In contrast, LCs induce strong responses after viral TLR-3 triggering (49). Thus,
LCs are more likely to respond to viral micro-organisms, while no or low responses
are observed upon encounter with (commensal) bacteria. Together these data
suggest that LCs selectively respond to micro-organisms, which depends on the
nature of the microbe.
For defense against fungal pathogens, DCs express high levels of dectin-1.
A main characteristic of dectin-1 is that TLR triggering is not required to induce
cytokine responses (11), although TLR-2 ligation has a collaborative effect on
yeast-induced activating signals (50). Triggering of dectin-1 on DCs by β-glucans
results in IL-12p70, IL-1β, IL-6 and IL-23 production to generate both Th1 and Th17
immunity, which are important for the clearance of invading fungal pathogens (51).
LCs express low amounts of dectin-1, but can recognize fungal β-glucans as well
as mannan via Langerin (chapter 2). Currently, it remains to be determined what
the immunological outcome of fungal interaction with LCs via Langerin is. Based on
the similarities between Langerin and DC-SIGN and the observation that Langerin
ligand mannan does not induce LC maturation or cytokine production, it is likely that
Langerin needs TLR triggering to modulate immune responses. Monocyte-derived
LCs strongly respond to ligands for TLR-2, which is involved in fungal recognition
and this induces IL-10 secretion (52). Thus, hypothetically, synergistic triggering
of TLRs and Langerin on LCs could result in a tailored immune response against
fungi, similar to the response observed by DC-SIGN and dectin-1. However, how
Langerin discriminates between pathogenic and opportunistic fungi, remains to be
determined. Possibly Langerin signaling is altered based on different carbohydrate
structures present on different fungi or on different fungal stages such as hyphae
versus yeast form. Carbohydrate-specifi c signaling is also a possibility as shown for
LCs and DCs represent two distinct DC subsets. LCs are more quiescent under
steady state conditions while DCs exhibit a more activated and mature phenotype
(53). This appears to be an intrinsic difference although the micro-environment and
location can further infl uence this. As sentinels lining the largest and most exposed
interface with the environment, which is colonized with many commensal bacteria
and fungi, there is more emphasis on the immune regulatory role and innate defense
against viruses for LCs. Noteworthy, other immune cells and receptors also contribute
to the induction of a proper immune response and therefore results should always be
placed in the correct context.
Part 2: Langerin+ LCs and DC-SIGN+ DCs in HIV-1 acquisition
HIV-1 transmission can occur through the gastro-intestinal mucosa, via
mother-to-child transmission, blood or plasma transfusion, shared-needle use and
sharp-incidences, but the majority of HIV-1 transmission occurs via heterosexual
transmission. At these anatomical sites LCs and DCs are present and in this section
their role in HIV-1 infection and transmission is discussed.
2.1 Langerin+ LCs versus DC-SIGN+ DCs
The role of DCs in transmission has been extensively studied using monocyte-
derived DCs. DCs facilitate HIV-1 transmission through several mechanisms in vitro,
which are both infection-mediated and independent of DC infection (54). Productive
infection of DCs leads to de novo production of virions, which subsequently infect
target cells. In addition, independent of DC infection, cell surface attachment of HIV-
1 to DCs results in the formation of an infectious synapse between DCs and T cells
through which HIV-1 is transferred to T cells. In both mechanisms, capture of HIV-1
can be enhanced by attachment of HIV-1 via cell surface receptors DC-SIGN and
Syndecan-3 (23;55). Moreover, HIV-1 transmission might also take place through
an exocytic pathway that involves HIV-1 associated exosomes: endocytosed HIV-
1 is incorporated into multivesicular bodies, which are rich in tetraspanins and
subsequently HIV-1 is transferred to T cells by exosomes (56). Furthermore, HIV-1
transmission can occur when HIV-1 is captured by DCs and internalized via the
endolysosomal pathway and subsequently directed towards the DCs-T cell synapse
(54). It is likely that these mechanisms exist next to each other and involve several
receptors, such as DC-SIGN and Syndecan-3 (23;55). Whether DC-SIGN+ DCs play
a role in in vivo HIV-1 transmission remains to be elucidated. Rectal mucosa is rich in
DC-SIGN+ DCs (57) where direct HIV-1 interaction with DC-SIGN+ DCs takes place
and it is suggested that in the absence of abrasions in the female genital tract, HIV-1
can reach DC-SIGN+ DCs by transcytosis through or between epithelial cells (58).
For a long time, HIV-1 transmission by LCs and DCs was thought to be mediated by
similar mechanisms. However, the function of Langerin is distinct from DC-SIGN and
this suggests different outcomes after HIV-1 encountering.
In vivo and ex vivo data support a role for LC infection in HIV-1 transmission (59-
61). Both the infection of LCs and the ability of LCs to transmit HIV-1 to T cells have
been demonstrated to occur ex vivo and in primate models (59-63). However, high
viral loads are needed to induce LC infection. In addition, sub-epithelial DCs are more
effi cient at transmitting HIV-1 to T cells compared to their epidermal counterparts
(59). Therefore, there appears to be a barrier that needs to be overcome to infect
As discussed previously, Langerin on LCs captures HIV-1 for targeting of the virus
to Birbeck granules for degradation, thereby protecting LCs against HIV-1 infection
(1;2). Since LCs line the squamous stratifi ed epithelium, they form a fi rst line of
defense against HIV-1 infection. The protective function of LCs may account for
the relatively low infectiousness of HIV-1 and the need for high inoculum in order to
achieve infection of LCs. However, Langerin function is abrogated by the use of high
viral loads, by saturating Langerin using antibodies or by inhibiting Langerin function
by carbohydrates. This suggests that LCs have a dual role in HIV-1 transmission,
depending on the activation state and the function of Langerin (65;66). This is
highlighted by the effect of micro-organisms causing sexually transmitted infections
(STIs) in HIV-1 acquisition, which is discussed in the next section.
2.2 Sexually transmitted infections
Epidemiological studies have demonstrated that the presence of STIs enhances
the risk of acquiring HIV-1 (67-70). Since LCs are the fi rst antigen presenting cells to
encounter invading pathogens, they could play a role in HIV-1 acquisition. Several
micro-organisms interact with Langerin, which might result in competition with HIV-1
for Langerin binding and subsequently enhances HIV-1 infection of LCs via CD4/
CCR5. HSV-2 is the most common viral STI and is a ligand for Langerin (chapter
4). These data suggest that in the presence of HSV-2, HIV-1 has to compete with
HSV-2 for Langerin binding. Saturation of Langerin by HSV-2 could subsequently
lead to infection of LCs by HIV-1 via CD4/CCR5. Similarly, C. albicans, which causes
vaginal candidiasis, also interacts with Langerin. Therefore, these data suggest that
C. albicans increases the risk of HIV-1 infection through a similar mechanism. HSV-2
has an opposite effect on DC-SIGN-mediated transmission. HSV-2 binding to DC-
SIGN and DC-SIGN down-regulation upon DC maturation by HSV-2 decreases HIV-
1 transmission (de Jong, unpublished observation). This suggests that Langerin and
DC-SIGN play opposing roles in the susceptibility to HIV-1 and that competition of
micro-organisms with HIV-1 for Langerin might facilitate HIV-1 infection.
Micro-organisms not only interact with LCs via Langerin, but also directly activate
LCs through TLRs. LCs residing the mucosa have an immature phenotype, which
include high expression of Langerin. Interactions with pathogens induce maturation
and subsequently down-regulation of Langerin from the cell surface. It has been
demonstrated that HSV-2 and TLR-3 ligand Poly(I:C) induce maturation of LCs
including a down-regulation of Langerin (chapter 4). Notably, both TLR-3-stimulated
LCs and HSV-2 infected LCs capture less HIV-1 gp120 via Langerin, suggesting
that these cells are more susceptible to HIV-1 infection and transmission. HSV-2
not only decreases Langerin function but also competes with HIV-1 for Langerin
binding. In addition, both HSV-2- and TLR-3-mediated LC maturation might also
induce a variety of phenotypical and transcriptional changes and subsequently affect
HIV-1 infection, replication and transmission. TLR-2 ligation via either heat-killed
L. monocytogenes or peptidoglycan (PGN) increases surface expression of CD4
in monocyte-derived LCs. In addition, post-entry restriction factor APOBEG3G is
reduced after TLR-2 stimulation with heat-killed L. monocytogenes or Pam3CSK4,
suggesting that Gram-positive bacteria enhance productive HIV-1 infection in LCs
by decreasing APOBEC3G (71). Thus, Langerin function and the protective barrier
of LCs can be breached by different mechanisms, including competition for Langerin
and the induction of LC activation by micro-organisms.
TLR ligands also affect transmission of HIV-1 independent of infection. In chapter
3 we have demonstrated that activation of primary human LCs through TLR-1/2
ligand Pam3CSK4 enhances HIV-1 transmission to target cells via increased HIV-1
capture. Pam3CSK4-mediated LC activation completely circumvents the protective
effect of Langerin by increasing HIV-1 capture and subsequent transmission. The
observed transmission is mainly mediated by cell-surface bound viral particles, since
transmission is abrogated in the presence of neutralizing anti-HIV-1 antibodies (65).
However, specifi c intracellular compartments in close contact with the cell-surface
might also be involved, since mature DCs transmit HIV-1 via specialized intracellular
compartments that are fully accessible for HIV-1 envelope specifi c inhibitors (72).
Together, these data indicate that LC activation by Pam3CSK4, induces or activates
receptors that have a high affi nity for HIV-1 compared to Langerin, since even at low
viral concentrations HIV-1 transmission is enhanced in the presence of Pam3CSK4.
However, the molecular mechanism involved remains to be determined. Activation
of CD34-derived LC-like cells with LPS and TNF-α increases HIV-1 transmission
independent of infection by internalization of HIV-1 in CD1a-containing multivesicular
bodies (66). These data suggest that in LCs HIV-1 is protected from degradation in
intracellular compartments and effi ciently transmitted upon interaction with target
cells through trans-infection, similar to mechanisms observed by DC-SIGN+ DCs.
STIs induce the production of pro-infl ammatory cytokines, which can affect the
susceptibility of LCs to HIV-1 transmission (65). As demonstrated in chapter 3, both
C. albicans and Neisseria gonorrhoea induce production of TNF-α in skin and vaginal
tissue (65). TNF-α induces HIV-1 replication of LCs ex vivo thereby increasing
HIV-1 transmission to CD4+ T cells (65). TNF-α has been shown to enhance HIV-
1 replication through activation of the transcription factor NF-B that increases
HIV-1 transcription (73;74). In agreement with these data, TNF-α enhances HIV-1
replication in LCs, which increases HIV-1 transmission to T cells due to enhanced
de novo HIV-1 production by LCs (65). However, it is important to note that although
TNF-α enhances replication, it does not increase HIV-1 entry into cells. Therefore,
the observed TNF-mediated increased transmission is dependent on Langerin
function and is only detected when Langerin is inhibited or saturated by high viral
concentrations (65). TNF-α is induced in genital mucosal fl uids from women with
bacterial vaginosis (75). These data strongly suggest that STIs induce TNF, which
enhances HIV-1 replication in LCs, and HIV-1 transmission to T cells (65). It has been
demonstrated that TNF-α exerts a similar effect on DC-SIGN+ DCs (76). Cytokines
induced by STIs can also regulate the expression and function of HIV-1 co-receptor
CXCR4. On DCs, CXCR4 expression is increased by IL-4 and TGFβ, and inhibited
by IFN-α,-β and -γ, while CCR5 expression on DCs is not susceptible to cytokines
(77). In addition to invasive STIs, sexual intercourse and (non-ulcerating) STIs can
induce (micro) abrasions in the epithelium. This can induce infl ammatory cytokine
production, such as TNF-α, in the epithelium as a reaction to trauma, thereby forming
a risk factor for HIV-1 acquisition.
Overall, HIV-1 infection and transmission by DC-SIGN+ DCs and Langerin+
LCs can be modulated at different levels. A fundamental difference exists between
Langerin and DC-SIGN in HIV-1 degradation and transmission under non-
infl ammatory conditions. In contrast to DC-SIGN, Langerin has not been shown
to enhance HIV-1 infection. The protective barrier formed by LCs is breached by
several mechanisms, which are both Langerin-dependent and -independent (Table
2). Most of the mechanisms have been explored ex vivo, often with epidermal LCs
or LC-like cells and monocyte-derived DCs, and further studies are necessary to
investigate the involvement of these mechanisms in vivo.
Table 2. A role for LCs in HIV-1 acquisition
HIV-1 degradation via
Langerin on LCs
no HIV-1 infection
Infection of LCs
de novo HIV-1 production
High viral load
Infection of LCs
de novo HIV-1 production
Fungi or HSV-2-mediated
competition for Langerin
Decrease in Langerin expression,
phenotypical/ transcriptional changes
as a result of LC maturation
Infection of LCs
de novo HIV-1 production
Up-regulation of HIV-1 entry receptors,
decrease in restriction factors
Enhanced HIV-1 entry,
enhanced de novo HIV-1
production and release
CD4 and APOBEC3G
Enhanced HIV-1 binding to LCs
Trans-infection of target cells
Infl ammation-induced TNF-α
Enhanced HIV-1 replication
2.3 Other players in the game?
There is a gap in our knowledge about the initial events during HIV-1 acquisition,
including the mechanisms of how HIV-1 enters the body and what cells are the
primary target cells in the mucosa (78). LCs lining the mucosal tissue account for
1-3% of the total cell population and therefore other cells can also infl uence HIV-1
acquisition. Mucosal LCs are surrounded by mainly epithelial cells and these genital
epithelial cells express proteins that mediate attachment of HIV-1 (79). However,
whether epithelial cells themselves are productively infected with HIV-1 remains
controversial (80;81). Likewise, the region at the interface of the endocervix and
ectocervix is rich in CD4+ T cells. Therefore, these cells can provide a route of entry
for HIV-1 (58). Furthermore, cell-conjugates of LCs and T cells provide a mechanism
of HIV-1 transfer (59;82).
The lamina propria of ectocervix and the dermis contain not only DCs but also
macrophages (83;84). These vaginal macrophages constitute approximately 10%
of the total number of leukocytes in these tissues and support HIV-1 replication
(85). In addition, other immune cells such as NK cells and pDCs play a role in the
innate defense against HIV-1 (58). Together the cells in tissues approached by HIV-1
create a specifi c micro-environment by not only interacting directly with HIV-1 and
other types of cells but also by producing an array of soluble factors such cytokines,
defensins, interferons, and mucins, which can further affect HIV-1 acquisition by
different mechanisms. None of the available research models completely mimic the
human HIV-1 exposed tissues, and it is therefore a future challenge to unravel the
events occurring in this specifi c micro-environment.
2.4 Host-defense mechanisms
Several biological factors infl uence the susceptibility to acquire and transmit HIV-
1. These include viral load, viral strain variants and different soluble factors (86;87).
This section discusses the effect of different soluble factors in human milk, semen
and body fl uids, the effect of the composition of the female and male genital tract and
the results of genetic alterations in HIV-1 acquisition and transmission.
Semen contains a variety of products that affect HIV-1 transmission. Spermatozoa
themselves interact with heparan sulfates on DCs, thereby facilitating HIV-1
transmission (88). In addition, seminal fl uid affects the genital tissue by neutralizing
the acidic environment, which could prolong survival of HIV-1 in the female genital
tract. Several soluble factors in semen have an effect on HIV-1 susceptibility; anti-
leukoprotease Secretory Leukocyte Protease Inhibitor (SLPI) present in saliva and
semen is able to protect CD4+ T cells against HIV-1 infection (89), while Semen-
derived Enhancers of Virus Infection (SEVI) captures HIV-1 virions and enhances
attachment of HIV-1 to T cells and macrophages (87) and Mucin-6, from seminal
plasma, enhances DC-mediated HIV-1 transmission by interacting with DC-SIGN
(90). It remains to be determined what the effects of semen are on LC-mediated
In addition to semen, human milk components possess a high level of anti-HIV-1
activity; lactoferrin and bile salt-stimulated lipase inhibit HIV-1 infection (91;92). In
chapter 6 MUC-1, from human milk, has been demonstrated to interact with DC-
SIGN thereby preventing DC-mediated HIV-1 transmission. Postnatal mother-to-
child transmission is mediated via human milk, and in the gastro-intestinal tract
DC-SIGN+ DCs are present, while Langerhans cells are absent. Therefore, MUC-1
provides a protective factor in human milk against HIV-1 transmission. It is likely
that different components of semen or breast milk have opposing effects on HIV-1
transmission, and therefore the net effect on HIV-1 infection is dependent on many
variables. Increased understanding of the factors involved may provide insight in
The composition of female genital tissue also affects the susceptibility to HIV-
1 infection. In general, HIV-1 infection occurs through the endocervix as well as
the multi-layered squamous epithelium of the vagina and ectocervix. Although the
endocervix consists of a single-layer columnar epithelium, it is covered with mucus,
which provides a protective barrier. LCs are expressed throughout the female genital
areas, but the density of the LCs in the female tissue, such as the cervix, is low
compared to male tissues, such as foreskin or glans penis (93). This may account
for the higher male-female transmission than female-male transmission. In general,
the female vagina is of low pH and commensal bacteria, such as Lactobacillus,
have a microbicidal effect that prevent HIV-1 acquisition (94;95). When this milieu
is altered, for example due to the colonization by pathogenic micro-organisms, an
environment can arise that promotes HIV-1 infection. In addition, the female genital
tissue is under continuous infl uence of hormones. During the menstrual cycle there
is a constant increase and decrease of the female epithelium; estrogen thickens
while progesterone thins the epithelium (96). Although the density of the cells is not
altered during menstrual cycle (95), the net number of LCs acting as a protective
barrier will be lower when the epithelium is thinner and the epithelium will be more
prone to damage. Furthermore, the expression of HIV-1 receptors varies during the
menstrual cycle (97). In addition, during ovulation hydration and alkalization of the
mucus occurs, which possibly decreases its barrier function (58).
Male circumcision has been demonstrated to decrease HIV-1 infection (98). The
outer foreskin and the glans penis are protected by a keratinized epithelium, which
act as a strong mechanical barrier, whereas the inner foreskin is poorly keratinized,
which increases vulnerability to HIV-1 infection (58). In addition, in non-circumcised
men the area between the foreskin and glans may provide a receptive environment
for STIs. These STIs might affect the protective barrier against HIV-1 transmission
by LCs localized in the foreskin, thereby greatly increasing HIV-1 susceptibility
(65). Epidemiological studies have demonstrated that circumcision prevents the
acquisition of STIs, such as HSV-2 and HPV (99).
Host genetic predispositions can further affect the susceptibility to HIV-1 infection
and include variation in the functionality and expression of CCR5, Langerin and DC-
SIGN. Individuals with a mutation in HIV-1 co-receptor CCR5, a homozygous 32 nt
deletion, are protected from primary HIV-1 infection (100). These data support the
observation that HIV-1 transmission occurs primarily by R5-tropic strains. Changes
in the function or binding affi nity of Langerin due to genetic alterations could also
affect the susceptibility to HIV-1 infection. Several polymorphisms in the Langerin
gene might affect the binding activity or stability (101). Clinical correlations between
mutations in the carbohydrate recognition domain of Langerin and HIV-1 susceptibility
have recently been identifi ed and preliminary data suggest that protection of LCs
against HIV-1 is affected by mutations in the carbohydrate recognition domain (M.
van der Vlist, unpublished publications), which decrease HIV-1 binding. In contrast,
the carbohydrate recognition domain of DC-SIGN contains no polymorphisms that
affect amino acid sequence, suggesting that DC-SIGN has been maintained under
a strong selective pressure that prevents accumulation of any amino acid changes
(102). On the other hand, a low number of repeat units in the neck region of DC-SIGN
is associated with a decreased risk of HIV-1 acquisition (103). This is likely to be due
to the reduced stability or reduced expression of DC-SIGN (104). Finally, variation
in responsiveness of human epidermal tissue to TLR ligands in combination with
HIV-1 susceptibility exists (65). Hence, this suggests that genetic variation in CCR5,
Langerin, DC-SIGN, and TLR expression play a role in the susceptibility to HIV-1
2.5 Microbicides in HIV-1 prevention: future prospectives
To date, no curative treatment or protective vaccine against HIV-1 is available,
while the HIV-1 pandemic is still expanding world-wide. Therefore, alternative
protective measures, such as the development of topically applied HIV-1 microbicides
in the form of genital creams, are needed to put a hold to HIV-1 acquisition (105).
These protective measures include microbicidal creams, and compounds targeting
HIV-1 attachment, entry, reverse transcriptase and integration. LeX (chapter 5) and
mermaid (chapter 7) exemplify the use of compounds to interfere with DC-SIGN-
mediated HIV-1 transmission.
The studies described in this thesis investigate the role of LCs and DCs in the
acquisition and transmission of HIV-1 through mucosal genital tissue. Acting as a
delicate barrier against HIV-1 by capturing HIV-1 through Langerin, it is essential to
maintain the LC barrier function, while it is favourable to inhibit DC-SIGN binding to
HIV-1 to prevent HIV-1 infection and transmission. Microbicides or mucosal vaccines
should prevent direct and indirect activation of LCs. LC activation can lead to HIV-
1 acquisition by lowering the viral threshold needed to induce direct LC infection,
by increasing HIV-1 replication and allowing trans-infection of target cells. Along
the same line, the fact that LCs do not respond well to certain bacterial products
could be an advantage. In addition, vaginal creams could possibly cause epithelial
disruptions and infl ammation at cellular levels (106), which in turn could actually
enhance HIV-1 infection. Therefore, the safest mucosal microbicide would be one
that is non-irritating, does not interfere with host-defense mechanisms and at the
same time does not induce viral resistance. Hence, in the rational design of new
topical microbicides it is important to carefully determine the effects at all levels.
Although DC-SIGN targeting appears to be a favorable target to decrease HIV-1
transmission, DC-SIGN is most likely also involved in innate immune processes that
are benefi cial for humans. Therefore, blocking DC-SIGN could also have adverse
effects. Appropriate models to investigate the interaction of microbicides with LCs or
DCs are a prerequisite.
In chapter 5, it is demonstrated that LCs derived from a Mutz-3 cell-line (Mu-
LCs) are a valuable tool to screen microbicides in their interaction with Langerin.
Mu-LCs and DCs resemble their in vivo counterparts and Mu-DCs are functional in
inducing anti-tumor T cell immunity (107;108). In addition, Mu-DCs are a valuable
tool as an unlimited source of DCs in cytotoxic T lymphocyte priming and lymph-
node homing capabilities (108;109). Although Mu-LCs are useful to screen potential
microbicides in Langerin-mediated HIV-1 transmission, a drawback is that this cell-
line exhibits a semi-mature phenotype and upon TLR stimulation produces a limited
amount of cytokines (110). Other LC-like cells have been generated from CD34+
cord blood (111) and blood monocytes (112). Like Mu-LCs these cells have a LC-
like phenotype and express Langerin. However, it is important to keep in mind their
limitations. These LC-like cells can express receptors that are not normally found on
primary LCs, such as the C-type lectins DC-SIGN. Moreover, some of these LC-like
cells have a different TLR expression profi le compared to primary LCs, which might
confound certain results obtained with these cells. Therefore, it is necessary to verify
obtained results with primary LCs. Since genital tissue is not readily available for
research purposes, skin obtained from breast or abdominal reconstructive surgery
are the most closely related models used as source of primary research material.
Noteworthy, human skin tissue is composed of a keratinized layer, which is absent in
the female genital tissues. Nevertheless, research using primary human material is
scarce, but very valuable since LCs are maintained in their physiological environment.
Monocyte-derived DCs have provided a great model to study the function of
DC-SIGN+ CD1a+ DCs in virology and immunology. However, questions remain
whether these cells resemble merely one specifi c DCs subset in vivo. Identifi cation
of immune cells in the dermis and sup-epithelium has been hampered by the lack of
distinct markers and it is thought that monocyte-derived DCs exhibited a phenotype
that combines several dermal and sup-epithelial subsets in vivo.
In the identifi cation of potential microbicides, microbicide transmission and
sterilization assays may be very valuable (113). Furthermore, attempts are made to
design skin equivalents in the laboratory by adding different cell(line)s into a matrix,
which is a promising tool to investigate the interaction between pathogens and the
different cell types (114). However, it will be a challenge to incorporate all primary cell
subsets into these systems.
Although the development of mouse models for HIV-1 infection has made great
improvements over the past few years (115-117), Rhesus macaque monkeys are
still the most often used in vivo model to study HIV-1 transmission. Primate studies
are laborious, relatively expensive and minimalized for ethical reasons. Therefore,
hormone treatments and high viral loads are often used to facilitate HIV-1 infection.
These conditions do not necessarily resemble HIV-1 transmission in humans which
is rather ineffi cient, therefore making it diffi cult to translate research between models.
In addition, interspecies differences should not be underestimated. Overall, each
method has its limitations, which should be carefully weighed against the advantages.
The development of innovative strategies against HIV-1 infection is essential in
the fi ght against HIV-1. Promising compounds are currently tested in phase III trials
(118). However, it is still a long way for the wide-spread distribution of functional
microbicides, especially to those who have limited access to healthcare. Therefore,
prevention and early treatment of STIs, education, advocacy for safe sex and
condom-use, and male circumcision are currently the main protective measures
against HIV-1 and other STIs. Knowledge attained from all levels of research can
contribute the understanding of HIV-1 infection, transmission and pathology and
ideally will lead to the development of a preventive HIV-1 vaccine.
Figure 2. Thesis outline: LC and DC interaction with pathogens. (A) Interaction with HIV-1. In intact
epithelium, LCs form a protective barrier against HIV-1 infection. Several conditions might affect the
protective function of LCs and induce HIV-1 transmission. Pro-infl ammatory cytokines or pathogenic
components induced by STIs and infl ammation activate LCs, which upon HIV-1 exposure, lead to
enhanced HIV-1 replication in LCs (TNF-α) or increased capture and transmission of HIV-1 (Pam3CSK4)
(chapter 3). Viral STI HSV-2 competes with HIV-1 for Langerin binding and induces down-regulation of
Langerin expression. Viral TLR-3 agonist Poly(I:C) induces strong maturation of LCs, resulting in Langerin
down-regulation in addition to phenotypical/transcriptional changes. Both HSV-2 and Poly(I:C) enhance
HIV-1 infection of LCs and subsequent de novo HIV-1 production (chapter 4).
LCs were discovered over 200 years ago by Paul Langerhans, while only 10
years ago Langerin and DC-SIGN have been identifi ed. Whereas DC-SIGN research
has taken a big leap since the development of monocyte-derived DCs and the
function has been widely explored, Langerin research has lagged behind and thus
far Langerin has mainly been used as a marker to identify Langerhans cells.
The studies in this thesis describe the role of Langerin+ LCs and DC-SIGN+ DCs
in the innate defense against pathogens, with a focus on HIV-1. This thesis has
elaborated on the effect of viral, bacterial and fungal sexually transmitted infections
on LC function and subsequent susceptibility to HIV-1 infection and has identifi ed
potential compounds to interfere with DC-SIGN-mediated HIV-1 transmission.
Together this thesis provides knowledge and insight in the pathogen recognition of
Langerin and DC-SIGN and can contribute to new therapeutic strategies in microbial
infections and the development of effective microbicides and mucosal vaccines.
Breaches in the epithelium, induced by infl ammation, STIs or sexual intercourse, allow HIV-1 to interact
directly with DC-SIGN+ DCs in the sub-epithelium, resulting in effi cient HIV-1 transmission. MUC-1 and
mermaid (chapter 6 and 7) prevent binding of HIV-1 to DC-SIGN, thereby preventing HIV-1 transmission.
Infl ammation induces an infl ux of T cells that are susceptible to direct infected by HIV-1. LCs derived
from Mutz-3 cell line provide a valuable tool to screen potential microbicides (not depicted, chapter 5).
(B) Langerin is a receptor for fungal species Candida species, Malassezia furfur and Saccharomyces
cerevisiae (chapter 1). DCs mediate HSV infection and transmission through DC-SIGN (chapter 8). HBV
does not interact with DCs (chapter 9), and Salp15 binding to DC-SIGN prevents T cell activation needed
against B. burgdorferi infection (chapter 10). This fi gure is modifi ed from de Jong, Journal of Internal
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