1. 1 Regulatory Actions of TLR2 and TLR4 in Leishmania donovani Infection in the Liver 2 3 He

45 Pages·2013·0.9 MB·English

IAI Accepts, published online ahead of print on 15 April 2013
Infect. Immun. doi:10.1128/IAI.01468-12
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
1 Manuscript IAI01468-12 (revised) 1.
2 Regulatory Actions of TLR2 and TLR4 in Leishmania donovani Infection in the Liver
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4 Henry W. Murray*#
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5 Yunhua Zhang*
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6 Yan Zhang+ de
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7 Vanitha S. Raman++ om

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8 Steven G. Reed++ p
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9 Xiaojing Ma+ a
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10 Departments of *Medicine and +Immunology and Microbiology, Weill Cornell Medical r
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11 College, New York, NY 10065 and the ++Infectious Diseases Research Institute, Seattle, n

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13 #Corresponding Author: Henry W. Murray, M.D. Weill Cornell Medical College, 1300 0
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14 York Avenue, New York, NY 10065. Telephone: [phone], FAX: [phone] and
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15 E-mail: [email protected] u
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16 Running Title: TLR2 and TLR4 in Visceral Leishmaniaisis
17 Key Words: visceral leishmaniasis, Leishmania donovani, TLR2 and TLR4, granuloma
18 formation, pentavalent antimony
19 2.
20 Abstract
21 In livers of susceptible but self-curing C57BL/6 mice, intracellular Leishmania donovani
22 infection enhanced TLR4 and TLR2 gene expression. In the liver, infected TLR4-/- mice
23 showed reduced IFN-γ, TNF and iNOS mRNA expression, higher-level and slowly-
24 resolving infection, delayed granuloma formation and little response to low-dose
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25 chemotherapy; in serum, the ratio of IFN-γ:IL-10 activity was decreased by 50%. In w
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26 contrast, in TLR2-/- mice, control of liver infection, parasite killing and granuloma a
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27 assembly were accelerated and chemotherapy’s efficacy enhanced. In livers of infected d

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28 TLR2-/- mice, mRNA expression was not increased for inflammatory cytokines or iNOS m

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29 nor decreased for IL-10; however, the serum IFN-γ:IL-10 ratio was increased by 6.5-fold tp
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30 and minimal responses to IL-10 receptor blockade suggested downregulated IL-10. In ia
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31 established infection in wild-type mice, blockading TLR2 induced parasite killing and m
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32 triggering TLR4 strengthened resistance and promoted chemotherapy’s effect. Thus, in g
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33 experimental L. donovani infection in the liver, TLR4 signaling upregulates and TLR2 n

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34 signaling downregulates macrophage antileishmanial activity, making both receptors r
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35 potential therapeutic targets in visceral leishmaniasis for engagement (TLR4) or 2
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36 blockade (TLR2). 9

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37 u
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40 3.
41 In visceral leishmaniasis, resident macrophages in the liver, spleen and bone
42 marrow are targeted and support intracellular parasite replication. In experimental
43 infection in the liver, C57BL/6 (B6) and BALB/c mice show initial susceptibility to
44 Leishmania donovani, but acquire resistance, reduce parasite burdens and express a
45 near-cure phenotype (1,2). This outcome involves CD4, CD8 and NK T cells and
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46 monocytes, is regulated by multiple activating cytokines, in particular, IFN-γ (3), and is o
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47 accompanied by conversion of parasitized tissue foci into inflammatory granulomas (1- lo
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48 11). Within these structures, populated by recruited mononuclear cells (10,11), the e
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49 effects of IL-10 and other initial counterregulatory mechanisms (2,5,12-15) are o
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50 overshadowed, yielding a net result of macrophage activation and parasite killing (16).
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51 The same T cell-, cytokine- and macrophage-mediated mechanisms also enable host /
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52 responsiveness to conventional antileishmanial chemotherapy, pentavalent antimony s
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53 (Sb) (1-3,9,17,18). o
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54 In leishmaniasis, early host-parasite interaction and proper initiation of host n

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55 defense likely involves recognition by Toll-like receptors (TLR) expressed on multiple r
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56 leukocyte populations, including dendritic cells, macrophages and T cells (19-22). In 2
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57 inducing initial inflammatory mechanisms, signals transduced by TLRs also likely shape 9

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58 the subsequent acquired responses that regulate T cell- and cytokine-induced g
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59 macrophage activation; thus, TLR signaling is considered well-positioned to govern s
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60 outcome in intracellular Leishmania infection (19-22).
61 4.
62 With two exceptions (20,23), however, information supporting a role for TLR
63 signaling in experimental visceral infection caused by L. donovani or L. infantum (L.
64 chagasi) has either been generated in vitro (19-22,24) or inferred indirectly from in vivo
65 observations. The latter have shown that expression of TLR2 and TLR4 mRNA in the
66 spleen correlates with parasite load in L. infantum-infected BALB/c mice (25), and that
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67 prophylactic and/or therapeutic injections of TLR2 (26,27), TLR4 (28) or TLR9 agonists o
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68 (29-31) enhance resistance in L. donovani-infected hamsters or BALB/c mice. Directly- lo
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69 generated in vivo information is limited. L. donovani-infected TLR7-/- B6 mice e
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70 demonstrate increased liver parasite burdens (23), while L. infantum-infected TLR2-/- B6 ro
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71 mice show accelerated parasite killing in draining lymph nodes following intraperitoneal
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72 inoculation (20) -- observations indicating an activating effect for TLR7 (23) and a /
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73 deactivating effect for TLR2 signaling (20). Our finding that L. donovani infection in the s
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74 liver provoked gene expression of TLR2 and TLR4 led us to test antileishmanial o
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75 responses in mice deficient in TLR2 or TLR4. o
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76 Materials and Methods r
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77 Animals and liver infection. TLR2-/- and TLR4-/- mice (B6 background), 0
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78 originally developed by Dr. S. Akira (Osaka University, Osaka, Japan), and IL-12p40-/- b
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79 and IFN-γ-/- B6 mice (Jackson Laboratories, Bar Harbor, ME) were bred at Weill Cornell u
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80 Medical College. B6 and BALB/c wild-type (WT) mice were purchased (Jackson
81 Laboratories). Groups of 3-5 female mice, aged 6-12 weeks, were injected via the tail
82 5.
83 vein with 1.5 x 107 hamster spleen homogenate-derived L. donovani amastigotes (LV9
84 strain). Endotoxin was not detected (< 0.1 EU/ml) in hamster spleen homogenates (LAL
85 Chromogenic Endotoxin Quantitation Kit, Pierce Biotechnology, Rockford, IL). Infection
86 was assessed microscopically using Giemsa-stained liver imprints in which parasite
87 burdens were measured by blinded counting of the number of amastigotes per 500 cell
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88 nuclei x organ weight (mg) (Leishman-Donovan units (LDU)) (1). Differences between o
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89 mean values were analyzed by a two-tailed Student’s t test. These studies were lo
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90 approved by the Medical College’s Institutional Animal Care and Use Committee. e
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91 Gene and mRNA expression in liver tissue. A high-density oligonucleotide m

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92 microarray system, Murine Genome U74A Array version 2 containing 12,488 genes tp
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93 (Affymetrix, Santa Clara, CA), was used to test liver tissue from uninfected and infected ia
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94 WT and IFN-γ-/- B6 mice. Total RNA was isolated from freshly obtained tissue, and m
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95 samples from 4 mice from each group were pooled. Performance of the microarray g
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96 experiment and the associated data analyses were carried out as previously described n

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97 (32). r
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98 For quantitative real-time RT-PCR testing, total RNA was isolated from liver 0
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99 tissue from individual mice using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and b
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100 reverse-transcribed into cDNA. RT-PCR was performed in an ABI 7400 System using u
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101 the SYBR GREEN PCR kit. GAPDH mRNA expression was used as the control for
102 quantitative analysis. The primers for detected genes are listed in Table S1. PCR
103 6.
104 cycling conditions were as follows: initial incubation step of 2 min at 500C, reverse
105 transcription of 60 min at 600C and 940C for 2 min, followed by 40 cycles of 15 sec at
106 950C for denaturation and 2 min at 620C for annealing and extension. To calculate
107 relative RNA levels, we used the formula: 2^(-ΔCt) where ΔCt = Ct (specific gene) – Ct
108 (GAPDH) (32).
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109 Tissue granuloma responses. The histologic response to infection was w
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110 evaluated microscopically in liver sections stained with hematoxylin and eosin. The a
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111 number of granulomas (infected Kupffer cells which had attracted > 5 mononuclear cells d

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112 (14,15)) was counted in 100 consecutive 40x fields and, at 100 parasitized foci, the m

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113 granulomatous reaction was scored as none, developing, mature and/or parasite-free tp
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114 (14). Mature granulomas show a core of fused parasitized Kupffer cells, numerous ia
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115 surrounding mononuclear cells and epitheloid-type changes (10). m
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116 Treatments. Groups of 3-4 mice were injected with: (a) pentavalent antimony /
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117 (Sb) (sodium stibogluconate, Pentostam, Wellcome Foundation Ltd., London, UK) IP in A
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118 0.2 ml of saline once on day +14 using an optimal leishmanicidal (500 mg/kg) or a lower il
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119 leishmanistatic dose (50 mg/kg) (14,15), (b) glucopyranosyl lipid A (GLA), a TLR4 0
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120 agonist (33), 20 ug in 0.1 ml of a stable emulsion (GLA-SE) (33) or 0.1 ml of SE alone b
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121 subcutaneously (SC) in the thigh on days +12 and +16, (c) Pam3CSK4 (InvivoGen, San u
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122 Diego, CA), a synthetic triacylated lipopeptide TLR1/2 agonist (27,34,35), 100 ug IP in
123 0.2 ml of saline on day +7 or day +14 or on days -2 and +7, (d) anti-TLR2 mAb (clone
124 7.
125 203325, R&D Systems, Minneapolis, MN) (36,37) or rat IgG, 50 ug IP in 0.2 ml of saline
126 once on day +14, or (e) anti-IL-10 receptor mAb (1B1.3A, Merck Research Labs, Palo
127 Alto, CA) or rat IgG, 0.2 mg IP in 0.2 ml of saline once on day +7 (14). Liver parasite
128 burdens were determined 7 days after the last treatment. At this time (day +14 or +21),
129 parasite killing or inhibition of replication, separate measures of antileishmanial activity
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130 (38), were calculated as reported (12,14,38) and indicated in the Table 1 legend. o
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131 IFN-γ and IL-10 protein determination. Serum was assayed for IFN-γ and IL-10 a
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132 activity using ELISA kits from BD Biosciences Pharmingen (San Diego, CA) and d

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133 BioLegend (San Diego, CA), respectively. Mean values were used to calculate the ratio m

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134 of IFN-γ:IL-10 serum activity. IL-10 activity was also measured in supernatants of tp
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135 triplicate cultures of WT and TLR2-/- spleen cells (5 x 106 cells/ml) stimulated for 24h ia
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136 with 10 ug/ml of Pam3CSK4 (39). m
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137 Results /
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138 L. donovani infection enhances expression of TLR2 and TLR4. Microarray p
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139 gene expression analysis using liver tissue from uninfected vs. infected WT mice ,

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140 indicated enhanced expression of TLR1, TLR2, TLR4 and TLR13 at both week 2 and 1
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141 week 3 after infection. These findings were confirmed by RT-PCR testing (Figure 1) y

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142 which demonstrated the following fold-increases in mRNA expression in 3-week e
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143 infected mice vs. mean values in uninfected controls: TLR1 (24.1), TLR2 (13.7), TLR4
144 (7.1) and TLR13 (12.8). TLR1 associates with TLR2 as a heterodimer to enable TLR2
145 signaling
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147 (34,40); the effects of TLR13 signaling are largely unknown (41).
148 Contrasting roles of TLR2 and TLR4. Three assays – kinetics of parasite
149 replication and outcome of infection, granuloma assembly and maturation at
150 parasitizedtissue foci, and responsiveness to chemotherapy (12,14) – were used to test
151 the behavior of L. donovani in livers of TLR-deficient mice. As shown in Figures 2-4,
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152 Figure S1 and in Table 1, WT mice (a) controlled (week 4) and then largely resolved w
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153 infection (week 8), (b) generated a granulomatous reaction which was evident at week a
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154 2, well-expressed at week 4 and receding by week 8, and (c) responded to optimal-dose d

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155 pentavalent Sb (500 mg/kg) with > 90% killing of liver amastigotes. Each of these three m

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156 T cell-dependent antileishmanial responses is initially regulated by both Th1- and Th2- tp
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157 type cytokines; after week 4, macrophage activation, intracellular parasite killing within ia
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158 mature granulomas and subsequent near-cure of liver infection is primarily Th1 cell- m
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159 dependent (1-11,16-18,38). g
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160 (i) Outcome of infection. As judged by altered liver parasite burdens (Figure 2), A
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161 both TLR2 and TLR4 were active in visceral infection; the signals transduced, however, il
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162 produced quite different outcomes. The effect of deficient TLR2 was pronounced, 0
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163 expressed early (week 2) and sustained; halted parasite replication followed by b
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164 accelerated amastigote killing assigned TLR2 a suppressive (deactivating) role. In u
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165 contrast, results in TLR4-/- mice indicated an activating role for TLR4, albeit modest, not t
166 discernible until infection was fully-established (week 4) and transient. Liver burdens in
167 9.
168 TLR4-/- mice exceeded those in WT mice at weeks 8 and 12 (p <.05); however, most
169 parasites had been killed by week 12, indicating a delayed, TLR4-independent
170 mechanism for macrophage activation.
171 (ii) Granuloma assembly. In livers of both groups of TLR-deficient mice, the
172 generation of functional granulomas, the tissue correlate of the successful inflammatory
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173 response to L. donovani (10,11), paralleled the capacity to control infection (Figures 3, 4 w
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174 and S1). In TLR2-/- mice, granuloma assembly, maturation and function (% parasite- a
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175 free) were accelerated -- at week 2, nearly all infected foci consisted of either d

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176 developing or mature granulomas; at week 4, 82-89% of granulomas were mature- m

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177 appearing and parasite-free. This upregulated reaction was not uncontrolled; by week tp
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178 8, most granulomas were either involuting or had resolved, indicating that TLR2 was not ia
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179 required to terminate the tissue inflammatory response. In contrast, TLR4-/- mice m
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180 showed little cellular reaction at most infected liver foci at week 2, developing and some g
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181 mature but parasite-filled granulomas at week 4, and while inflammatory, mature n

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182 granulomas were fully-established by week 8, few were microscopically parasite-free r
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183 until week 12 (not shown). 2
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184 (iii) Response to Sb chemotherapy. Since the mechanisms that control parasite b
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185 replication and granuloma assembly in the L. donovani-infected liver also enable Sb’s u
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186 antileishmanial efficacy (1-3,9,17,19,38), we expected Sb’s effect in liver infection to be
187 reduced in TLR4-/- and enhanced in TLR2-/- mice. However, intracellular parasite killing
188 10.
189 after treatment with optimal-dose Sb (500 mg/kg) was intact in TLR4-/- mice (Table 1).
190 Nevertheless, the anticipated responses were uncovered by using suboptimal Sb (50
191 mg/kg), a more subtle test for regulatory effects on chemotherapy’s efficacy (12). Low-
192 dose Sb produced leishmanistatic activity in WT mice (81% inhibition of parasite
193 replication), no effect in TLR4-/- mice (2% inhibition) and enhanced parasite killing in
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194 TLR2-/- mice beyond that already in-progress in untreated mice. o
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195 Antileishmanial defense mechanisms in livers of TLR-deficient mice. The a
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196 divergent kinetics of infection in livers of TLR-deficient mice suggested upregulated d

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197 activating and/or downregulated suppressive mechanisms in TLR2-/- mice and the m

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198 opposite in TLR4-/- mice. Therefore, using liver tissue from 3-week infected animals, we tp
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199 first focused on mRNA expression of (a) cytokines that direct activating Th1-type ia
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200 responses and/or macrophage activation (e.g., IL-12, IFN-γ, TNF (3,38,42-44)), (b) m
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201 cytokines that extinguish these responses (e.g., IL-10, IL-27 (12,13)), and (c) iNOS, the g
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202 mechanism primarily used by the IFN-γ-activated macrophage to kill L. donovani (16). n

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203 For IFN-γ and IL-10, we also assayed protein levels in serum and calculated the ratio of r
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204 IFN-γ:IL-10 activity. 2
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205 (i) mRNA expression in infected liver. In TLR4-/- mice, there was no change in b
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206 relative mRNA expression (< 2-fold difference vs. WT mean values) of IL-12p35, IL- u
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207 12p40, IL-10 or IL-27 (not shown); mean IFN-γ, TNF and iNOS expression was 2.1- to t
208 6.1-fold lower (Figure 5). TLR2-/- mice, however, showed no change (< 2-fold
209 11.