Chapter 4 Discussion
4.6 Potential role of macrophages in the GC reaction to influence the generation of
target for innovative adjuvants in vaccine development and the development of
cross-protective effects of vaccines. On the flip side, PPAR-δ inactivation might be a
prospective therapy toward autoimmune diseases. Our ongoing research aims to unravel
the molecular mechanism of immunomodulatory effects of GW501516 and PPAR-δ
activation both in vitro and in vivo by using conditional and inducible knockout mice of
FcγRIIB gene and to modulate GC response using various candidate compounds.
4.6 Potential role of macrophages in the GC reaction to influence the generation of Ag-specific Abs
It has been reported that defective phagocytosis of macrophages can lead to
autoimmunity (Fond and Ravichandran, 2016). FcγRIIB can block IC-mediated
activation of FcγR and other activating receptors in macrophages. Moreover,
overexpression of FcγRIIB in myeloid cells suppresses host immunity against bacterial
infection. Conversely, FcγRIIB-deficient macrophages increase their phagocytic
property (Brownlie, et al., 2008). The gene transcription of FcγRIIB has been shown to
be down-regulated in a macrophage cell line by PPAR-δ agonist (Adhikary et al., 2015).
45
It is then crucial to determine whether the absence of FcγRIIB gene can promote
macrophage function in vivo.
Splenic macrophage subpopulations are diverse and their functions remain elusive.
The unique tingible body macrophages (TBMs; Mer+DNaseI+) are predominantly
scattered in the GC. TBMs express Mer receptor tyrosine kinase, which mediates
phagocytic activity and regulates cytokine production (Rahman, 2011). They
phagocytose apoptotic cells and thereby contain condensed chromatin fragments. TBMs
are thought to play a role in down-regulation of GC reaction (Rahman, 2011). The
marginal zone (MZ) surrounds lymphoid follicles, where MZ macrophages (MZMs;
MARCO+CD169+) and metallophilic macrophages (MMMs; MOMA+) are abundantly
present (McGaha and Karlsson, 2016). MZMs are specialized macrophages that
phagocytosed apoptotic materials entering the spleen from circulation to minimize the
immunogenicity of autoantigens (McGaha et al., 2011). MMMs distribute adjacent to
the T- and B-cell-rich zones. Interestingly, MZMs and MMMs disappear when B cells
were absent before birth or gradually depleted after birth, suggesting a critical role of B
cells in the maintenance of splenic MZ structure (Nolte, et al., 2004). Interaction with
46
MZ B cells is also crucial for efficient homing of MZMs and MMMs and for efficient
removal of blood-borne pathogens coming into the MZ.
To the best of our knowledge, whether the fate of GC B cells during affinity
maturation is influenced by macrophages, whether FcγRIIB regulates phagocytic
activity of GC and MZ macrophages, and how macrophages might interact with B cells
in the GC are all incompletely understood. We have recently generated conditional
knockout mice of FcγRIIB gene in myeloid cells to investigate the potentially important
role of macrophages in the selection of GC B cells. Moreover, this mouse strain will
help explain and conclude our findings on the effects of GW501516 of macrophages
during affinity maturation of GC reaction in secondary immunization.
47
Figures
48
Figure 1
Mouse immunization schedules.(A)
(B)
(C)
49
Figure 1. Mouse immunization schedules.
(A~C) 7-to-10-week-old female mice were primarily immunized with 50 µg of
NP20-CGG admixed with 50µl of alum intraperitoneally on day 1. The second booster
was given on day 28 with the same antigen and route. The “x” denotes the day of
collecting serum samples. Before the second booster, serum samples were collected on
days 14 and 27. (A) Comparison of wild-type and FcγRIIB232T/T mice. Mice were
sacrificed on day 35 after primary immunization. Sera were collected on day 14, day 28
and day 35. No second booster was given. (B) C57BL/6 mice were daily given nilotinib
at 2 mg/kg/day or vehicle intraperitoneally from the sixth day to the ninth day after the
second booster. Mice were sacrificed on the tenth day after the second booster (day 38).
Serum was collected on day 37. (C) C57BL/6 mice were daily given vehicle,
GW501516 3 or 6 mg/kg/day intraperitoneally from the sixth day to the ninth day after
the second booster. Mice were sacrificed on the tenth day after the second booster (day
38). Serum samples were collected on day 37.
50
Figure 2
Analysis of the differences of affinity maturation status between wild-type and FcγRIIB232T/T mice over time.
.
Figure 2. Analysis of the differences of affinity maturation status between
wild-type and FcγRIIB 232T/T mice over time.
On days 14, 28 and 35 after the primary immunization with NP-CGG, sera were
collected. The serum NP-specific IgG levels were analyzed by ELISA assay. The status
of affinity maturation was measured by the OD450 ratio of serum NP7-specific
IgG/NP30-specific IgGs between wild-type (n=3) and FcγRIIB232T/T (n=4) mice on day
14 (P=0.0243), day 28 (P=0.0089) and day 35 (P=0.0095).
51
Figure 3
Analysis of the differences of affinity maturation status between wild-type and FcγRIIB232T/T mice over time.
52
Figure 3. Compare FcγRIIB232T/T mutant and wild type mice of FcγRIIB cluster in
the lipid raft with confocal microscopy.
(A) Confocal image (400x magnification) of B-lymphocyte retrieved from the spleens
8-week-old FcγRIIB232T/T mutant and wild type mice. FITC-labeled cholera toxin B
binds to the GM1 ganglioside of the lipid raft and display green fluorescence.
Cy3-labeled anti-rabbit IgG binds to the anti-mouse IgM and display red fluorescence.
FcγRIIB stabilized in the lipid raft would display yellow fluorescence. In wild type
mice, cap structures were formed (arrows) at 30 to 60 min but FcγRIIB232T/T mutant
mice did not show much merged yellow fluorescence (arrow head). (B) Quantitative
representation of FcγRIIB localized in the lipid raft with Metamorph analysis tool.
FcγRIIB232T/T mutant mice showed significant impaired lipid raft colocalization over 15
min (P < 0.05).
53
Figure 4
Analysis of the effect of nilotinib on the affinity maturation of female wild-type mice.
54
Figure 4. Analysis of the effect of nilotinib on the affinity maturation of female
wild-type mice.
(A, B) Wild type female mice were given nilotinib 2 mg/kg/day or vehicle given from
the seventh day to the ninth day after the second booster. (A) Analysis of IgG secreting
PC numbers with ELISPOT assay. Number of low affinity NP-specific IgG secreting
PCs per 2.4 x 104 splenocytes by subtracting the NP7 specific IgG-secreting PCs from
NP30 specific IgG-secreting PCs. Comparisons between vehicle (n=6) and
nilotinib-treated group (n=8) (P=0.0087). (B) Analysis of serum NP-specific IgG
concentration by ELISA assay. The affinity maturation was measured by the OD450
ratio of serum NP7-specific IgG/NP30-specific IgG between vehicle (n=5) and
nilotinib-treated group (n=5) (P=0.0283).
55
Figure 5
Analyzing IgG-secreting PCs in the spleen of female C57BL/6 mice immunized with two different GW501516 doses by ELISPOT assay.(A)
(B)
56
immunized with two different GW501516 doses by ELISPOT assay.
(A, B) ELISPOT quantification (upper panel) and demonstrative graph (lower panel) of
the high-affinity and total NP-specific IgG PCs per 3 x 105 splenocytes. Female
C57BL/6 mice, 10 days after the second booster (day 39). Vehicle (n=6, as control
group), GW501516 3 mg/kg/day (n=9), GW501516 6 mg/kg/day (n=7) were
administered, respectively, during the sixth to the ninth day after the second booster. (A)
High-affinity NP7-specific IgG-secreting PC count: GW501516 3 mg/kg/day (ns), 6
57
mg/kg/day (ns). (B) Total NP30-specific IgG-secreting PC count: GW501516 3
mg/kg/day (P=0.040), 6 mg/kg/day (P=0.049), compared with the control group.
(C) Low-affinity NP-specific IgG secreting PCs measure by subtracting the NP7
specific IgG-secreting PCs from NP30 specific IgG-secreting cells. Low-affinity
NP-specific IgG secreting PC count: GW501516 3 mg/kg/day (P=0.042), 6 mg/kg/day
(P=0.005). Results were analyzed with unpaired, two-tailed student t test and shown as
mean SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, no significance
58
Figure 6
Measurement of serum IgG levels of female C57BL/6 mice immunized with two different GW501516 doses by ELISA assay.(A)
(B)
(C)
59
F
Figure 6. Measurement of serum IgG levels of female C57BL/6 mice immunized
with two different GW501516 doses by ELISA assay.
(A, B) Serum high-affinity and total NP-specific IgGs in the serum of female C57BL/6
mice, 10 days after the second booster (day 39). Vehicle (n=5, as control group),
GW501516 3 mg/kg/day (n=6), GW501516 6 mg/kg/day (n=6) were administered,
respectively, during the sixth to the ninth day after the second booster. Serum was
diluted in 4 x104 folds. (A) Serum high-affinity NP2-specific IgG: GW501516
3mg/kg/day (ns), 6mg/kg/day (ns). (B) Serum total NP30-specific IgG of treatment with
GW501516 3 mg/kg/day (P=0.037) or 6 mg/kg/day (P=0.025), compared with the
control group. (C) The affinity maturation measurement by dividing the NP2 specific
IgG serum level by the NP30 specific IgG serum levels. NP2/NP30: GW501516 3
mg/kg/day (P=0.035), 6 mg/kg/day (ns). Results were analyzed with unpaired,
two-tailed student t test and shown as mean SEM. *P < 0.05, **P < 0.01, ***P <
0.001 and ns, no significance. AU denotes arbitrary units
60
Figure 7
Analyzing IgM-secreting PCs in the spleen of female C57BL/6 mice immunized with two different GW501516 doses by ELISPOT assay(A)
(B)
61
(C)
Figure 7. Analyzing IgM-secreting PCs in the spleen of female C57BL/6 mice
immunized with two different GW501516 doses by ELISPOT assay
(A, B) ELISPOT quantification (upper panel) and demonstrative graph (lower panel) of
high-affinity and total NP-specific IgM-secreting cells per 3x105 splenocytes of female
C57BL/6 mice, 10 days after the second booster (day 39). Vehicle (n=7, as control
group), GW501516 3 mg/kg/day (n=10), GW501516 6 mg/kg/day (n=8) were
administered, respectively, during the sixth to the ninth day after the second booster. (A)
High-affinity NP7-specific IgM-secreting PC count: GW501516 3 mg/kg/day (ns),
62
6mg/kg/day (ns). (B) Total NP30-specific IgM-secreting PC count: GW501516 3
mg/kg/day (ns), 6 mg/kg/day (P=0.017), compared with the control group.
(C) Low-affinity NP-specific IgM secreting PCs measure by subtracting the NP7
specific IgM-secreting PCs from NP30 specific IgM-secreting PCs. Low-affinity
NP-specific IgM secreting PC count: GW501516 3 mg/kg/day (P=0.45), 6 mg/kg/day
(P=0.0469) Results were analyzed with unpaired, two-tailed student t test and shown as
mean SEM,*P < 0.05, **P < 0.01, ***P < 0.001 and ns, no significance.
63
Figure 8
Measurement of the serum IgM levels of female C57BL/6 mice immunized with two different GW501516 doses by ELISA assay(A)
(B)
(C)
y
64
Figure 8. Measurement of the serum IgM levels of female C57BL/6 mice
immunized with two different GW501516 doses by ELISA assay.
(A, B) Serum high-affinity and total NP-specific IgMs in the serum of female C57BL/6
mice, 10 days after the second booster (day 39). Vehicle (n=5, as control group),
GW501516 3 mg/kg/day (n=5), GW501516 6 mg/kg/day (n=5) were administered,
respectively, during the sixth to the ninth day after the second booster. Serum was
diluted 2 x 104 times. (A) Serum high-affinity NP2-specific IgGs: GW501516 3
mg/kg/day (P=0.046), 6 mg/kg/day (P=0.013). (B) Serum total NP30-specific IgMs.
GW501516 3 mg/kg/day (P=0.14), 6 mg/kg/day (P=0.045), compared with the control
group. (C) Affinity maturation measured by dividing the NP2 specific IgM serum levels
by the NP30 specific IgM serum levels. NP2/NP30: GW501516 3 mg/kg/day (P=0.03),
6 mg/kg/day (P=0.019). Results were analyzed with unpaired, two-tailed student t test
and shown as mean SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, no
significance. AU denotes arbitrary units.
65
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Appendix
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Publication List
1. Jhou JP, Yu IS, Hwai H, Chen CS, Chen PL, Tzeng SJ*. The lupus-associated Fcγ receptor IIb-I232T polymorphism results in impairment in the negative selection of low-affinity germinal center B cells via c-Abl in mice. Arthritis and Rheumatology.
2018 Nov;70(11):1866-1878. doi: 10.1002/art.40555.
2. Hwai W, Chen YY, Tzeng SJ*. B-cell ELISpot assay to quantify antigen-specific antibody-secreting cells in human peripheral mononuclear cells. In the 3rd edition of Handbook of ELISPOT in Springer/Humana Press book series “Methods in Molecular Biology” 2018;1808:133−141. doi: 10.1007/978-1-4939-8567-8_11.
3. Tseng TC, Huang DY, Lai LC, Hwai H, Hsiao YW, Jhou JP, Chuang EY, Tzeng SJ*. Dual immuno-renal targeting of 7-benzylidenenaltrexone alleviates lupus nephritis via FcγRIIB and HO-1. Journal of Molecular Medicine (Berlin) 2018 May;96(5):413-425. doi:
10.1007/s00109-018-1626-73
Publications
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The lupus-associated FcγRIIB-I232T polymorphism results in impairment in the negative selection of low-affinity germinal center B cells via c-Abl in mice
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B-cell ELISpot assay to quantify antigen-specific antibody-secreting cells in human peripheral blood mononuclear cells
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Dual immuno-renal targeting of 7-benzylidenenaltrexone alleviates lupus nephritis via FcγRIIB and HO-1
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