142 J. Carrisoza-Urbina et al.
(NO) released in response to M. bovis were sig-
nificantly increased compared to levels released
by uninfected cells. Macrophage activation by
the classical pathway induced large NO release;
however, lipopolysaccharide (LPS) pretreatment
followed by M. bovis infection showed a higher
NO production. Under these conditions M. bovis
replication was significantly decreased. Neutral-
izing NO production with nG-monomethyl-l-
arginine monoacetate (MMLA) in M. bovis and
LPS treatments confirmed that macrophage
microbicidal activity against M. bovis was NO
dependent. These results demonstrated that NO
generation is a key process in anti-mycobacterial
activity in bovine macrophages (Esquivel-Solís
et al., 2013).
Macrophages induced to acquire an alter-
native activation status by incubation with IL-4
decreased basal production of NO induced by
LPS alone or in combination with INF-γ. Short-
term incubation (4 hours) with IL-4 showed an
increase in the number of bacilli phagocytized,
independently of the strain virulence. The viru-
lent strain played an important role in phago-
cytosis inducing a higher number of bacilli
phagocytized in comparison with BCG; however,
this number was affected in those treatments
where IL-4 incubation was maintained for 24
hours, reducing the phagocytosis levels with no
differences between bacterial strains. This effect
was more evident when a virulent M. bovis strain
was used. The number of intracellular survival
bacilli phagocytized showed a higher intracellu-
lar proliferation in cells incubated for 4 hours
with IL-4; however, when the stimulation was
maintained for 24 hours, macrophages further
reduced their microbicidal ability. LPS alone or
in combination with M. bovis infection increased
pro-inflammatory cytokine gene expression of
bovine macrophages, whereas IL-4 treatment
reversed the effect of classical activation by
decreasing mRNA levels of the inflammatory
mediators. Collectively this body of evidence
indicates that macrophage alternative activa-
tion induced functional changes that resulted in
modification of the rate of phagocytosis,
decreased NO production and iNOS mRNA lev-
els, and as a consequence increased pathogen
intracellular survival suggesting that alterna-
tively activated macrophages are in general
more permissive to M. bovis growth (Castillo-
Velázquez et al., 2011).
10.3.2 Dendritic cellsDCs are specialized cells in antigen presentation,
capable of activating different cells of the
immune system such as NK, T γd cells and naive
T cells that are important for the initiation and
maintenance of the immune response (Fabrik
et al., 2013; Pearce and Everts, 2015). DCs, just
like macrophages, express PRRs that serve for
bacterial recognition. Hence, once DCs phagocy-
tize bacilli, antigens are processed and uploaded
into the major histocompatibility complex
(MHC) molecules for presentation in the lymph
nodes (Hope et al., 2004).
Mycobacterial interaction with DCs may
exert a dual role in innate immune response
modulation. For instance, the M. bovis manno-
sylated lipoarabinomannan (ManLAM) is recog-
nized by the DC-SIGN receptor (Hope et al.,
2004; Fabrik et al., 2013; Stamm et al., 2015),
which induces the expression of the potent
immunosuppressant IL-10, affecting the anti-
gen presentation process by inhibiting the
migration and maturation of DCs (Hope et al.,
2004; Fabrik et al., 2013). In this way, the onset
of the immune response is delayed and is insuf-
ficient to eradicate mycobacteria. On the other
hand, the interplay between DCs and mycobac-
teria increases the expression of DC surface mol-
ecules such as MHC-II, CD80, CD86 and CD40,
that leads to T cells activation in an attempt to
eliminate the bacterial invader (Hope et al.,
2004; Pearce and Everts, 2015).
Indeed, mycobacteria have the ability to
modulate the cytokine profile of DCs. Dendritic
cell infection with M. tuberculosis or M. bovis
BCG has been associated with a higher expres-
sion of IL-12, TNF-α, IL-1 and IL-6 (Hope et al.,
2004), which are essential in the control of
tuberculosis. IL-12 participates in the acquired
immune response through increasing IFN-γ and
TNF-α secretion by T cells; this, in turn, increases
the microbicidal activity of macrophages and
NK cells in order to destroy the bacilli (Hope
et al., 2004; Denis and Buddle, 2008).
It has also been demonstrated that DCs have
a higher capacity to phagocytize M. bovis; how-
ever, once internalized in these cells, M. bovis also
shows enhanced replication since the DCs release
between five and ten times lower amounts of NO,
IL-1β and TNF-α compared to macrophages. Sur-
vival and replication of mycobacteria within DCs