Background and rationale
Mononuclear phagocytes are an important target of HIV infection and replication.
However, the potential role of cytokine-directed polarization into either proinflammatory
(M1) or anti-inflammatory (M2) macrophages has not been
thoroughly investigated in the context of this viral disease. We have already
defined the fundamental features of human M1 and M2 macrophages by exposing
conventional monocyte-derived macrophages (MDM) to either tumor necrosis
factor-a plus interferon-g (M1) or interleukin-4 (IL-4; M2) in terms of differential
expression and modulation of cell surface determinants as well as of secretion of
chemokines and cytokines that may play a relevant role in HIV infection. Of
interest, both M1 and M2 polarization resulted in a transient decrease of CCR5-
dependent (R5) HIV-1 replication in comparison to control unpolarized autologous
MDM, an inhibitory pattern supported, at least in part, by a transient
downregulation of CD4, the primary viral receptor for entry, in contrast to the
viral co-receptor CCR5 that is not decreased in M1-MDM and it is even
upregulated under M2-polarizing conditions at the time of infection. Kinetic
analyses indicated that M1 polarization induces a stronger but less durable
inhibitory effect on HIV-1 replication compared to M2 conditioning. M1-MDM show
an increased secretion of the CCR5-binding chemokine CCL3 as well as of CXCL10
and IL-6. Conversely, M2-MDM are characterized by a modest up-regulation of
the HIV inhibitory chemokine CCL22 and of IL-10. Of note, M1 polarization
induces a delayed downregulation of M2-related chemokine and cytokines and
vice versa. These preliminary findings suggest that polarization of tissue
macrophages in either M1 or M2 cells in response to cytokines or bacterial
products may favor a state of latent over productive infection and, therefore,
impair the capacity of the adaptive T cells response to eliminate macrophage viral
reservoirs.
Specific goals
While the pro-inflammatory (M1) pathway of differentiation and activation of
macrophages is well characterized, there are different modalities to induce M2
polarization in addition to IL-4, including cell stimulation by IL-10 and Toll-like
receptor ligands whereas, in addition, deactivating factors such as transforming
growth factor-β can also profoundly affect macrophage function. The regulatory
effect of these cytokines on HIV infection and replication has been previously
investigated, but not in the context of macrophage polarization. Furthermore, the
possibility to exploit cell lines, such as promonocytic U937 cells, as inducible
models of M1/M2 polarization will be part of the project. In addition to learn how
to perform experiments under biosafety level-3 (BSL-3) conditions and the basic
fundamentals to study live HIV infections, this project will provide expertise on
the immunology of human macrophages, and on molecular assays related to
characterize viral transcription, protein expression, and living cell imaging.
Key references
E. Cassol, M. Alfano, P. Biswas, & G. Poli. Monocyte-derived macrophages and
myeloid cell lines as targets of HIV-1 replication and persistence. J. Leuk. Biol.
80:1018-1030, 2006.
E. Cassol, C. Rizzi, L. Cassetta, M. Alfano, & G. Poli. M1 and M2a polarization of
human monocyte-derived macrophages inhibits HIV-1 replication by distinct
mechanisms. J. Immunol., 182: 6237-6246, 2009.
E. Cassol, L. Cassetta, C. Rizzi, M. Alfano, & G. Poli. Macrophage polarization and
HIV-1 infection. J. Leuk. Biol., 87:599-608, 2010.

Background and rationale
The definition of the precise modality of HIV infection via interaction with a
primary cell surface receptor (CD4) and chemokine co-receptor (CCR5 or CXCR4)
has allowed to characterize both the relevance of these two co-receptors in interindividual
viral transmission (both horizontal, sexual or blood-related, and
vertical, i.e. mother-to-child) and in the world pandemics. It is very clear that
both events are driven mostly, if not exclusively, by usage of CCR5 (R5 virus)
whereas the utilization of CXCR4 occurs only during the late stages of HIV disease
and it is usually associated with the maintenance of CCR5 use (so called
dualtropic R5X4 strains). Thus, R5 viruses have an evolutionary advantage vs.
CXCR4-using viruses in terms of efficiency of transmission and spreading in the
general population. In this regard, we have early described a model system based
on the suboptimal stimulation of cord blood derived primary cell lines in which R5
viruses efficiently replicate while X4 do not unless re-stimulated by T cell
activating signals (E. Vicenzi et al., J. Virol. 1999).
Based on this system, we have defined a transcriptome of about 2,000
genes either commonly or differentially regulated by molecularly isogenic HIV-1
clones differing only for their chemokine co-receptor use (CCR5 or CXCR4).
Specific goals
In order to investigate which genes were differentially affected by isogenic
R5 and X4 HIV-1 by a microarray approach we have recently introduced a restimulation
step with anti-CD3 + anti-CD28 mAbs and confirmed the previously
described pattern of R5-skewed replication.
In addition, in collaboration with Prof. Alessandro Aiuti (TIGET) we have
recently reproduced a similarly discordant phenotype based on post-entry
restriction of X4 viruses in peripheral blood leukocytes isolated from children
rather than adults; in the same setting, we have also explored the capacity of
leukocytes isolated from children affected by congenital immunodeficiency either
before and after having received gene therapy-based replacement of their CD34+
cells, with similar results (S.A. Mariani et al., in preparation).
Therefore, the full phenotypic and functional characterization of both the
originally modified and of this second model system will represent the central aim
of the project and will include the comparison of gene expression profiles
determined in the original cord blood based model described above.
Key references
E. Vicenzi, P. Panina-Bordignon, P. Biswas, A. Brambilla, M. Cota, F. Sinigaglia, &
G. Poli. Envelope-dependent restriction of human immunodeficiency virus type 1
spreading in CD4(+) T lymphocytes: R5 but not X4 viruses replicate in the
absence of T-cell receptor restimulation. J. Virol. 73:7515-7523, 1999.
E. Vicenzi, P. Panina-Bordignon, G. Vallanti, P. Di Lucia, & G. Poli. Restricted
replication of primary HIV-1 isolates using both CCR5 and CXCR4 in Th2 but not
in Th1 CD4+ T cells. J. Leuk. Biol., 72:913-920, 2002.
S.A. Mariani, E. Vicenzi, & G. Poli. Asymmetric HIV-1 co-receptor use in activated
CD4+ T lymphocytes. J. Transl. Res., 2010, in press.