impaired innate immunity in irak4 deficient mice leads to defective

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Impaired innate immunity in IRAK4 deficient mice leads to 1 defective Type 1 T cell responses, B cell expansion and 2

enhanced susceptibility to infection with Toxoplasma gondii 3 Running Title: IRAK4 mediates resistance to T. gondii 4

5 Samantha R. Béla1,2,*; Míriam S. Dutra1,2,*; Ernest Mui3; Alexandre Montpetit4; 6

Fernanda S. Oliveira2; Sérgio C. Oliveira2; Rosa M. E. Arantes2; Lis R. Antonelli1; 7 Rima McLeod3; and Ricardo T. Gazzinelli, RT1,2,5# 8

9 1Centro de Pesquisas René Rachou, CPqRR – Fundação Oswaldo Cruz, Avenida 10 Augusto de Lima 1517, Belo Horizonte 30190-002, MG, Brazil; 2Instituto de Ciências 11 Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Belo 12 Horizonte 31270-901, MG, Brazil; 3Departments of Surgery, Ophthalmology and 13 Pediatrics (Infectious Disease), University of Chicago, 5801 South Ellis Avenue, 14 Chicago 60637, IL, US; 4McGill University and Génome Québec Innovation Center, 15 740 Dr. Penfield Avenue, Montréal H3A 1A4, Québec, Canada; 5University of 16 Massachusetts Medical School, 364 Plantation St, Worcester 01605, MA, US. 17

Keywords: Cytokines, TLRs, innate immunity, IRAK4, Toxoplasma gondii 18 19 #Ricardo Gazzinelli, CPqRR/FIOCRUZ, Avenida Augusto de Lima 1517, Belo Horizonte 20 30190-002, MG, Brazil; +55-31-3349-7774; [email protected] . 21 *These Authors contributed equally to the current work. 22

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00328-12 IAI Accepts, published online ahead of print on 1 October 2012

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Abstract 23 IL1R-associated kinase (IRAK) 4 is a member of the IRAK family, and has an 24 important role in inducing the production of inflammatory mediators. This kinase is 25 downstream of MyD88, an adaptor protein essential for Toll-Like Receptors (TLRs) 26 function. Herein, we investigated the role of this kinase in IRAK4 deficient mice 27 orally infected with the cystogenic ME49 strain of T. gondii. IRAK4-/- mice displayed 28 higher morbidity, tissue parasitism and accelerated mortality, when compared to the 29 control mice. Lymphoid folicles and germinal centers from infected IRAK4-/- mice 30 were significantly smaller. Consistently, IRAK4-/- mice showed a defect on splenic B 31 cell activation and expansion as well as diminished production of IFNγ by T 32 lymphocytes. The myeloid compartment was also affected. Both frequency and 33 ability of dendritic cells (DCs) and monocytes/macrophage to produce IL-12 were 34 significantly decreased, and resistance to infection with Toxoplasma was rescued by 35 treating IRAK4-/- mice with rIL-12. Additionally, we report the association of IRAK4 36 haplotype-tagging single nucleotide polymorphisms (tag-SNPs) with congenital 37 toxoplasmosis in infected affected individuals (rs1461567 and rs4251513, p<0.023 38 and p<0.045, respectively). Thus, signaling via IRAK4 is essential for activation of 39 innate immune cells, development of parasite specific acquired immunity and host 40 resistance to infection with T. gondii. 41 42 43


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Introduction 44 Toxoplasma gondii is an ubiquitous intracellular protozoan parasite that infects a 45 broad range of vertebrate hosts, including humans and domestic animals (18). 46 Clinically, toxoplasmosis may be asymptomatic or present signs and symptoms that 47 vary depending on the host genetic background, immunological status (22, 49) and 48 the type of infective parasite (44). In immunocompetent host, Toxoplasma infection 49 is controlled by an effective immune response and causes little or no overt signs of 50 disease. However, in immunocompromised patients or during pregnancy, T. gondii 51 may emerge as a serious infection, which if not treated, often leads to lethality (52). 52 A hallmark of T. gondii infection is the induction of a strong Th1 mediated immunity, 53 especially with production of IFNγ that keeps in check the tachyzoites, the rapidly 54 multiplying form of the parasite. The control of tachyzoite replication allows the 55 development of the chronic and asymptomatic stage of disease, which is maintained 56 by the bradyzoite stage of the parasite (10). While the CD4+ T and CD8+ T cells are 57 critical in maintaining chronic toxoplasmosis, innate immunity seems to play an 58 important role in host resistance at early stages of infection. Activation of the cellular 59 compartment of the innate immune system triggers effector mechanisms that are 60 directly responsible for the control/elimination of the intracellular parasites. In 61 addition, cytokines produced by innate immune cells will direct the development of 62 acquired immunity, which is responsible for immune surveillance at later stages of 63 infection (10). 64 The activation of innate immune cells is achieved through engaging an array of germ 65 line-encoded pattern-recognition receptors to detect invariant pathogen motifs (51). 66 During infection with T. gondii the production of IL-12 by myeloid cells is induced by 67


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the activation of Toll-Like Receptors (TLRs), which triggers signaling through the 68 adapter protein MyD88 (9, 31, 43, 55). Following MyD88 association with TLRs, the 69 former recruits IL-1 receptor-associated kinase (IRAK) 1 (4), IRAK2 (4), IRAK M (53) 70 and IRAK4 (23, 46) to TLRs through interaction of the death domains (50) of both 71 molecules. Several recent studies using transgenic animals indicate that each IRAK 72 protein has a distinct function (47). Unlike IRAK1 or M, deficiency in IRAK4 leads to 73 serious flaw in the activation of transcription factors (NFκB, AP1, cJUN) and 74 production of several inflammatory cytokines induced by signaling through TLRs 75 (46). In humans, IRAK4 has 460 amino acids sharing 84% similarity with the murine 76 IRAK4 protein. The role of IRAK4 in the activation of the immune system was 77 demonstrated in a murine model of viral infection (47) and in humans who suffer 78 from recurrent infections with pyogenic bacteria (30, 35, 39). 79 In the present study, we observed both activation of innate immunity and 80 development of acquired immunity was highly compromised in IRAK4 deficient 81 mice. In particular, a defect on IL-12 production by myeloid cells and IFN-γ by T cells 82 was shown to be a primary mechanism responsible for enhanced susceptibility of 83 IRAK4-/- mice to infection with T. gondii. Finally, the relevance of our findings for 84 human disease is highlighted by our findings that two haplotype-tagging single 85 nucleotide polymorphisms (tag-SNPs) in the IRAK4 gene were significantly 86 associated with congenital toxoplasmosis in humans. 87 Material and Methods 88 Mice. IRAK4+/- mice (46) in the C57BL6 and 129 genetic background were kept as 89 heterozygote breeders to generate both homozygotes and heretozygotes mice used 90 in our studies. IRAK4-/-, IRAK+/- and 129/Ola mice were bred and maintained in 91


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microisolators at Federal University of Minas Gerais and René Rachou Research 92 Institute. Male mice between 8 and 12 weeks of age were used in all experiments. All 93 procedures were conducted according to institutional guidelines for animal ethics. 94 Antibodies. I-A/I-E-Biotin (2G9); I-A/I-E-APC (M5/114.15.2), CD86-PECy7 (IT2.2), 95 NK1.1-FITC (PK136), CD3-PerCP5.5 (145-2C11), CD19-FITC (1D3), CD69-PE 96 (H1.2F3), CD8-PECy7 (53-6.7), CD62L-APC (MEL-14), FC Block, IL-12-PE (C17.8), 97 TNFα-PECy7 (TN3-19), IFNγ-PE (XMG1.2) and Ki67-PerCP5.5 (20Raj1), from BD 98 Pharmingen; as well as: CD19-APC-eFluor780 (1D3), CD86-APC (B7-2), CD11c-99 Alexa700 (N418), CD40-PECy5 (1C10), GR1/Ly-6G and Ly-6C-PECy5 (RB6-8C5), CD4-100 PO (RM4-5), CD44-PB (IM7), CD11b-FITC (M1/70), F4/80-APC (BM8), and 101 recombinant IL-12 (rIL-12) from eBioscience; and streptavidin-QD605 (Q10101MP), 102 from Invitrogen. 103 Parasites, Soluble Toxoplasma Antigen, and experimental infections. Infective 104 cysts of the ME49 strain were obtained from brain tissue of Swiss mice at 30-40 days 105 post-infection. Soluble T. gondii antigen (STAg) was obtained from tachyzoites of the 106 RH strain of T. gondii, which is maintained in Swiss mice by passage at regular 107 periods of 48 to 72h. Briefly, a parasite suspension obtained from the peritoneal 108 cavity was sonicated, centrifuged, supernatant collected, and the protein content 109 determined by the Bradford method (15). For experiments, mice were infected 110 orally with 0.1 ml of brain homogenates adjusted to contain 20 cysts; and monitored 111 for mortality/morbidity for 30 days or euthanized at days 7, 9 or 20-30 days (for 112 cyst counts) after infection, when blood, brain, lung, liver and spleen were collected. 113 Morbidity was assessed based on the clinical parameters that were scored as 114 follows: bright and active (0); ruffled coat (1); 10% weight loss (2); hunched, 115


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tottering gait, starry stiff coat (3); reluctance to move and 20% weight loss (4); and 116 death (5). 117 In vivo treatment with Recombinant IL-12 (rIL-12). IRAK4-/- mice were 118 challenged per-oral with the ME-49 strain and treated daily with intraperitoneal 119 injections of rIL-12 (150 ng/mouse )(eBioscience), for 7 days, beginning on day 1 120 post-infection. As control, IRAK4-/- mice were infected and received PBS in an 121 identical schedule (40). 122 Quantitative real-time PCR (RT-PCR). Spleen and liver were harvested from mice 123 at 7 or 9 days after infection and frozen in liquid nitrogen. Total DNA was extracted 124 using phenol-chloroform protocol and quantified by UV spectrophotometry 125 (NanoDrop Spectrophotometer, Thermo Scientific). Quantitative PCR were setup in a 126 final volume of 20 µl containing 25 ng of total tissue DNA, 10 ng of each primer and 127 1x of the iQ SYBR Green Supermix (BioRad), and performed on a sequence detection 128 system (7000 Sequence Detection System, Applied Biosystems). The results are 129 expressed in pg of parasite DNA per 25 ng of tissue DNA. The primers used for 130 amplification of T. gondii B1 gene were: forward, 5’- CTGGCAAATACAGGTGAAATG-131 3’; and reverse, 5’- GTGTACTGCGAAAATGAATCC-3’ (31). 132 Histopathology. Brain, lung, liver and spleens were fixed with 10% buffered 133 formaldehyde and processed for paraffin embedding. Tissue sections were cut with a 134 microtome, and stained with hematoxylin and eosin or Giemsa. The histological 135 aspects of parenchyma tissue were evaluated for inflammation (endothelial 136 reactivity, perivascular cell infiltration and necrosis) and parasitism (tissue cysts) by 137 light microscopy. Morphometric aspects of the spleen as diameter of the follicle and 138 the germinal center were evaluated using the program KS 300 (Zeiss, Germany). 139


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Isolation of splenic leukocytes: Spleens from controls and infected mice were 140 incubated with 0.4 mg/ml Liberase CI solution (Roche Biochemicals) for 30 minutes 141 at 37°C. Single-cell suspensions were then passed through a 100 μm nylon cell 142 strainer. Erythrocytes were then lysed and splenocytes resuspended in RPMI 1640 143 medium supplemented with gentamicin sulfate 40 mg and 10% fetal bovine serum 144 (Gibco). Cells were then cultured for 48 h in 96 well plates in the presence or the 145 absence of exogenous stimuli for cytokine measurement and flow cytometer. 146 Cytokine measurements. Cytokine levels were assessed in supernatants of 147 splenocyte cultures and serum samples harvested from IRAK4-/- and IRAK4+/- mice 148 at 7 days post-infection with ME49 strain of T. gondii. IL-12p70 levels were 149 measured by enzyme-linked immunosorbent assay kit (eBioscience), and the IFNγ, 150 TNFα, IL-10, IL-6, MCP-1 levels were determined using the BD™ Cytometric Bead 151 Array Mouse Inflammation kit, according to manufacturers’ instructions. 152 Flow Cytometry. To analyze the phenotype of splenocyte subpopulations, cell 153 suspensions were stained with monoclonal antibodies specific. Two million freshly 154 isolated splenocytes were analyzed for surface markers expressed by DCs, 155 monocytes/macrophages, CD4, CD8, B and natural killer (NK) cells. To assess 156 cytokine production by DCs and macrophage/monocytes, splenic cells were cultured 157 in the presence of Brefeldin-A (GolgiPlug Protein Transport Inhibitor, BD 158 Biosciences, San Jose, CA) for 6 h, 37°C, 5% CO2, with LPS (100 ng/ml), STAg (5 159 µg/ml), or medium alone. To assess T lymphocytes and NK cells, 1x106 splenic cells 160 were cultured for 20 h in the presence of STAg (5 µg/ml), anti-CD3 (1 µg/ml) or 161 medium alone. Brefeldin-A was added in the cultures in the last 12 hours before 162 staining. To assess B cells, 1x106 splenic cells were analyzed without stimulus. Cell 163


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suspensions were then washed and stained with different combinations of the 164 antibodies described above. Cell suspensions were then washed, fixed and 165 permeabilized for the intracellular cytokine or proliferation marker staining (anti-166 IL-12p40, anti-TNFα, anti-IFNγ or Ki67), according to the manufacturer’s 167 instructions. Cells were finally suspended in 200 µl of 2% paraformaldehyde, data 168 collected in a LSR II with Diva (BD Biosciences) and analyzed with FlowJo (Tree Star 169 Inc., Ashland, OR) software v 8.8.6. 170 Families studied and their evaluations. Since 1981, persons with congenital 171 toxoplasmosis and their families participate in a longitudinal study of congenital 172 toxoplasmosis, which takes place across their lifetimes (27-29, 32, 36, 42). 173 Enrollment begins when children are first diagnosed, at birth (in some cases in 174 utero) or at the time of diagnosis. They are then re-examined at 1, 3.5, 5, 7.5, 10, 15 175 and 20+ years of age (27-29, 32, 36, 42). Serologic testing is performed in the 176 Remington US Reference laboratory at The Palo Alto Medical Foundation-Stanford 177 University or at the Institut de Puericulture in Paris, France (for US expatriate families 178 who returned from France to the US). General pediatric evaluation, ophthalmologic, 179 neurologic, hearing, developmental pediatric standardized testing according to a 180 pre-specified protocol, and hematologic examination were performed for every child 181 at birth and at each follow up visit. Full fundus retinal photographs, OCT and visual 182 fields were obtained for children who were at ages or cognitive capacity appropriate 183 for cooperation with such testing. A computerized tomographic brain scan was 184 obtained for every infant at birth or at the time of diagnosis for other congenitally 185 infected persons (27-29, 32, 36, 42). Of the 179 infected children who were 186 genotyped for this study, 124 (83%) had clinically confirmed brain calcifications 187 with/without hydrocephalus and/or retinal lesions at birth or time of diagnosis. This 188


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study was performed by the National Collaborative Chicago-based Congenital 189 Toxoplasmosis Study (NCCCTS) Group (27-29, 32, 36, 42). Physician and scientists 190 who conducted these evaluations in the NCCCTS are listed in the acknowledgements. 191 All procedures were performed in person and in accordance with Institutional and 192 NIH Guidelines and consistent with informed consent from parents or legal 193 guardians and adult congenitally infected persons. 194 Genotyping of Human IRAK4. Peripheral Blood Mononuclear Cells (PBMCs) from 195 179 children and their parent or parents’ were cryopreserved at -135 degrees until 196 placed in buffer for DNA extraction (38). This DNA was genotyped for 7 tag-SNPs 197 distributed throughout the IRAK4 gene. The tag-SNPs were selected from the 198 HapMap project (http://www.hapmap.org) using 10 kb flanking sequence on each 199 side of IRAK4 gene, a MAF cutoff of 5% in CEU and r2 threshold of 0.8. The Tagger 200 tool within Haploview (http://www.broad.mit.edu/mpg/haploview) was used to 201 select tag-SNPs. Allelic association analysis was performed for the 124 children with 202 confirmed clinical findings in the eye and/or brain] using UNPHASED 203 (http://www.mrc-bsu.cam.ac.uk/personal/frank/software/unphased). 204 Statistical Analysis: For the studies of mice, the cumulative mortality of experimental 205 groups was analyzed using Log-rank test. The difference between groups was 206 verified using t test or Mann–Whitney for parametric or non-parametric data, 207 respectively. All tests were performed using GraphPad Prism (v 5.0) (GraphPad 208 Softwares Inc., La Jolla CA), and differences were considered statistically significant 209 when p < 0.05. For genetic studies of humans, allelic association analysis was 210 performed using a conventional transmission disequilibrium test (TDT). Linkage 211 disequilibrium (LD) is non-random association of alleles at two or more loci. It is an 212


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approximation for existence of historical recombination between 2 loci. The p values 213 were calculated using Haploview (http://www.broadinstitute.org/haploview). p 214 values of 0.05 or less were considered significant for association with disease. 215 216 Results 217 Functional IRAK4 mediates the control of parasite replication, during primary 218 infection in mice. To evaluate the role of IRAK4 during T. gondii infection, IRAK4-/-, 219 IRAK4+/-, and the wild type (WT) 129 mice were orally infected with 20 cysts of the 220 ME49 strain. Animals were evaluated up to 30 days post-infection. Mortality of the 221 infected IRAK4-/- mice was detected as early as 10 days, reaching 100% by 30 days 222 post-infection. In contrast, no death was observed in either the heterozygous or 129 223 animals (Figure 1A). Heterozygote and WT mice showed signs of morbidity 224 classified as mild to moderate, which resolved by 2-3 weeks of infection. In contrast, 225 IRAK4-/- mice infected with T. gondii showed severe signs of morbidity, including 226 neurological symptoms indicating cerebral commitment and weight loss (Figure 1B 227 and 1C). Since the IRAK4-/- are in the C57BL/6 and 129/Ola genetic background and 228 not fully backcrossed, we used IRAK4+/- littermates as controls in most experiments, 229 as indicated in figure legends. Importantly, for all experiments that we used two 230 parallel groups, the parasitological and immunological tests were similar, when 231 comparing 129 and IRAK4+/- mice infected with T. gondii (data not shown). 232 Additionally, to assess whether the clinical signs of diseases and lethality in IRAK4-/- 233 mice were due to an uncontrolled parasite replication, we analyzed the tissue 234 parasitism in IRAK4-/-, IRAK4+/- and WT mice. Brains of animals infected for more 235 than 20 days were harvested, tissue sections were stained with Giemsa and cysts 236


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were quantified. Brains of IRAK4-/- mice infected with T. gondii showed significantly 237 higher cyst counts, when compared with the control groups (IRAK4+/- and 129) 238 (Figures 1D and 1E). Additionally, IRAK4-/- mice showed numerous foci of necrosis 239 in the central nervous system (CNS), always associated with parasitic cysts, while 240 the IRAK4+/- and 129 mice had few necrotic foci, which were not associated with 241 parasites. Nevertheless, heterozygote and 129 animals showed a moderate 242 inflammatory infiltrate with foci of perivascular inflammation, suggesting an 243 effective inflammatory response, which effectively controls parasite expansion in the 244 CNS of both IRAK4+/- and WT mice. 245 Spleen and liver were harvested 7 and 9 days post-infection for parasitism 246 evaluation by RT-PCR. T. gondii DNA levels were greater both in spleen and liver of 247 IRAK4-/- mice than in heterozygote (Figure 2E). These results indicate that control of 248 tachyzoite replication and tissue parasitism during primary infection, and therefore, 249 host resistance to T. gondii infection, are dependent on functional IRAK4. 250 Defective expansion of splenic B lymphocytes in IRAK4 deficient mice. When 251 examined at seven days after infection, spleens from IRAK4-/- mice showed an 252 altered structure. The area of follicles and germinal centers from infected IRAK4-/- 253 mice, but not in uninfected animals, were relatively smaller than IRAK4+/- mice, with 254 little cellular infiltration and scant lymphocytic halo (Figure 2A) suggesting that 255 both lymphoid and myeloid cells were less active in this organ during T. gondii 256 infection. As expected, total number of B cells was significantly lower in IRAK4-/- 257 mice, as compared to IRAK4+/- mice (Figure 2B - bar graphs). Consistently, the 258 frequency of activated B cells, as assessed by CD86 and CD69 expression, was 259 significantly decreased in IRAK4-/- mice, as compared to heterozygote mice infected 260


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with T. gondii (Figure 2C). To confirm that the observed changes in the germinal 261 centers were, at least in part, due to altered proliferation of B cells, we also analyzed 262 the expression of the cell cycle marker Ki67 (interphase stage). We observed that 263 upon infection with T. gondii, the number of Ki67 expressing B lymphocytes was 264 dramatically increased in IRAK4+/- mice, but not in the IRAK4-/- mice (Figure 2B). 265 Impaired production of pro-inflammatory cytokines in IRAK4 deficient mice 266 infected with T. gondii. Cytokines such as IL-12, IFNγ and TNFα play an important 267 role in host resistance to T. gondii infection. Thus, we assessed the cytokine 268 production by cells from IRAK4-/- mice infected with T. gondii. Sera were collected 269 from mice at 7 days post-infection. Spleens were also harvested and splenocytes 270 cultured for 48 h. IRAK4-/- animals infected with T. gondii displayed impaired IL-12, 271 IFNγ, and TNFα production. This lower level of cytokines was observed in both sera 272 (Figure 3A) and supernatants of splenocyte cultures (Figure 3B). The levels of IL-10 273 and MCP-1 (CCL2) were also significantly lower in the supernatant of cultures of 274 spleen cells derived from infected IRAK4-/-. 275 Altered distribution and impaired cytokine production by myeloid cells from 276 IRAK4 deficient challenged with T. gondii. We next asked whether the deficiency 277 of IRAK4 is associated with changes in myeloid effector cells compartment. To 278 investigate this, we assessed monocytes/macrophages subpopulations and DCs in 279 spleen of infected mice. Monocytes/macrophages subpopulations were defined by 280 the expression of F4/80, which is a specific marker for monocytes/macrophages, 281 and CD11b as well as GR1 (Figure 4A and 4B). Moreover, all these subpopulations 282 express MHC class II, which is not expressed by neutrophils. A very small population 283 (less then 1%) of neutrophils (F4/80-CD11b+GR1+MHCII-) is seen in spleens from 284


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mice infected with T. gondii. This was also confirmed by histological analysis of the 285 spleens from heterozygote and IRAK4 knockout mice (data not show). Notably, 286 IRAK4-/- mice displayed a lower frequency of F4/80+ cells in comparison with the 287 IRAK4+/- mice. The absolute number of all three different macrophage/monocyte 288 subsets, (F4/80hiCD11bloGr1lo, F4/80intCD11bintGr1lo, F4/80loCD11bhiGr1hi cells) 289 were decreased in the IRAK4-/- mice, when compared with the IRAK+/- mice infected 290 with T. gondii (Figure 4C). Importantly, the frequency of monocytes/macrophages 291 producing IL-12 or TNFα when stimulated with STAg was higher in IRAK4+/- mice 292 than in the IRAK4-/- animals (Figure 4C). Among the macrophages/monocytes, the 293 F4/80intCD11bintGr1lo cells were the main source of IL-12, followed by the 294 F4/80hiCD11bloGr1hi subset, whereas the production of TNFα was induced only in 295 the F4/80intCD11bintGr1lo subpopulation in the IRAK4+/- mice compared to 296 uninfected controls (Figure 4C). 297 Higher absolute numbers of CD11c+MHCIIhi cells were found in IRAK4+/- mice. Upon 298 T. gondii infection, IRAK4-/- mice displayed lower frequency of IL-12-producing DCs 299 even in the absence of STAg stimulation. Production of both IL-12 and TNF-α by DCs 300 was enhanced upon STAg stimulation (Figure 5A). In addition, when assessing the 301 expression of CD86 and CD40, the IRAK4 deficient mice showed decreased level of 302 both activation markers on DCs, as compared with heterozygote mice (Figure 5B). 303 Hence, our results indicate a failure on activation of DCs from IRAK4-/- mice infected 304 with T. gondii (Figure 5B). To evaluate the importance of the impairment on IL-12 305 production by DCs, IRAK4-/- infected with T. gondii were treated daily beginning at 306 day 1 post-infection. As expected, IRAK4-/- treated with rIL-12 become resistant to 307


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infection (Figure 5C) and all mice survived over 30 days after challenge with the 308 ME49 strain of T. gondii. 309 Impaired Th1-mediated immune responses in IRAK4 deficient mice infected 310 with T. gondii. Since the activation of antigen presenting cells was diminished in 311 IRAK4 deficient mice, we next asked whether the T cell responses were also 312 impaired. Our results showed that the total number of CD4+ T, but not CD8+ T 313 lymphocytes was significantly lower in the knockout, when compared to IRAK4+/- 314 mice (Figure 6A). Importantly, the frequencies of IFNγ producing CD4+ T as well as 315 CD8+ T lymphocytes was decreased in IRAK4-/- mice when compared to IRAK4+/- 316 mice upon STAg stimulation (Figure 6B). Altogether, our results indicate that the 317 decreased production of IL-12 by macrophages and DCs leads to a consequent 318 impairment in the IFNγ production by T cells, and it is likely to be the primary 319 mechanism responsible for enhanced susceptibility of IRAK4-/- mice to T. gondii 320 infection. 321 Human congenital toxoplasmosis associates with IRAK4 SNPs. In humans, 322 Congenital toxoplasmosis is a serious disease which threatens sight, cognition, and 323 life. It was therefore of interest to study whether there is an association of 324 congenital toxoplasmosis. Seven tag-SNPs in the IRAK4 gene were studied. The 7 325 SNPs were rs4251545, rs1057190, rs4251580, rs4251513, rs1461567, rs4251520, 326 rs17121283. The frequency of the SNP variants were tested in the North American 327 cohort of case-parent trios for 124 congenitally infected persons. We used an r2 328 threshold of 0.8. SNPs with a call rate >90% and in Hardy-Weinberg Equilibrium 329 (HWE) in parents were kept for further analysis. Among the tested SNPs, rs1461567 330 and rs4251513 associated with congenital toxoplasmosis (p < 0.023 and p < 0.045, 331 respectively) (Figure 7). An etiological variant of SNPs in strong LD with these 332


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markers could account for observed associations with susceptibility to this 333 congenital disease. 334 335 Discussion 336

Key features of infection with T. gondii are the strong activation of innate 337 immunity and the development of a solid antigen-specific T-cell mediated immune 338 responses (10). Among the innate immune receptors, TLRs are prominent in 339 recognition of protozoan molecular patterns (14). The activation of all TLRs (except 340 TLR3) sequentially triggers the adaptor molecule, MyD88; the downstream kinase, 341 IRAK4; and the transcription factor, nuclear factor κB (NF-κB) culminating in the 342 production of pro-inflammatory cytokines (46) that are essential for the 343 development of acquired immunity and host resistance to T. gondii. Hence, one of the 344 strongest data suggesting that TLRs are important in host resistance to 345 toxoplasmosis are the studies demonstrating that MyD88-/- mice have an impaired 346 production of IL-12 and IFNγ, enhanced parasitism and accelerated mortality (7, 9, 347 31, 43, 45). 348 Similar to the MyD88-/-, IRAK4-deficient mice had a dramatic increase of parasitism 349 in the spleen, liver, and brain, as well as accelerated mortality that were associated 350 with impaired production of the pro-inflammatory cytokines IL-12, TNF-α and IFN-351 γ. IRAK4 also mediate the function of IL-1 and IL-18 receptors. However, the IL-1R 352 and the IL-18 knockout mice are not more susceptible to T. gondii infection (19). 353 Thus, the most likely explanation for the impaired response of IRAK4-/- macrophages 354 is the disrupted signaling triggered by TLR exposed to T. gondii components. Indeed, 355 it was previously reported a defective release of nitrogen radical intermediates by 356


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IRAK4-/- macrophages exposed to T. gondii-derived the Heat Shock Protein 70 357 (HSP70), which is a TLR4 agonist (34). In addition, studies performed in our 358 laboratory show that glycosylphosphatidylinositol anchors extracted from T. gondii 359 tachyzoites activate TLR2 and TLR4 (9). However, deficiencies in TLR2 and TLR4 360 had no major impact on mouse resistance to T. gondii infection (9). On the other 361 hand, TLR11, the counterpart receptor for a profilin-like protein from T. gondii, was 362 shown to mediate IL-12 production by DCs, and its deficiency results in enhanced 363 susceptibility to infection (41, 55). TLR9 was also shown to mediate inflammation in 364 per-oral infection with T. gondii (33). Notably, we reported that 3d mice are 365 extremely susceptible to infection with T. gondii (31). The 3d mice have a missense 366 allele in UNC93B1 gene, which results in non-functional TLR3, TLR7, TLR9 and 367 TLR11 (6, 50). Thus, our results indicate that in addition of TLR11, the nucleic-acid 368 sensing TLRs have a fundamental role of in host resistance to Toxoplasma infection. 369 The activation of DCs through TLRs induces not only cytokine secretion, but also 370 enhances their capacity to prime naïve pathogen-specific T-cells, providing signals 371 (co-stimulatory molecules) that are important for the development of Th1 cells 372 during protozoan infection (14, 51). Consistently, in oppose to DCs from infected 373 heterozygote mice, the expression of co-stimulatory molecules (i.e., CD86 and CD40) 374 as well as the IL-12 was not augmented in IRAK4-/- mice. Our in vivo results contrast 375 with the ones obtained in vitro by Aosai and colleagues (2), who reported that DC 376 maturation induced by T. gondii-derived HSP70 was mediated by TLR4, but 377 independent of the MyD88 pathway. The defective DCs in infected IRAK4-/- mice is of 378 special interest, because impaired IL-12 production results in diminished IFNγ 379 production by T lymphocytes, and enhanced susceptibility to T. gondii (16, 25, 26, 380


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43, 48, 54). Indeed, treatment with rIL-12 rescued resistance of IRAK4 deficient mice 381 to experimental infection with T. gondii. 382 It was also noticeable that the frequency of monocytes/macrophages (CD11bhi 383 F4/80loGR1hi) that is increased in control mice infected with T. gondii, was not 384 observed in the spleen from IRAK4-/- mice. In addition to contribute as a source of IL-385 12, inflammatory monocytes are essential as effector cells for controlling parasite 386 replication in vivo (12). The decreased frequencies of monocytes/macrophages 387 observed in IRAK4-/- mice infected with T. gondii can be explained based on the 388 impaired production of MCP-1 (CCL2) by splenocytes from IRAK4 deficient mice, as 389 this chemokine is of critical importance for the recruitment of inflammatory 390 monocytes (1, 11). 391 IRAK4 deficiency is also associated with an altered function of T and B lymphocytes 392 (21, 39). As previously reported, upon infection with T. gondii the total number of T 393 cell subsets was decreased (3, 17, 24). This was observed in both heterozygote and 394 IRAK4-/- mice. Nevertheless, a major defect was observed in terms of IFN-γ 395 production by both CD4+ and CD8+ T cells from IRAK4 deficient mice. A likely 396 explanation for these findings is the impaired expression of co-stimulatory 397 molecules and IL-12 by dendritic cells from IRAK4-/- mice, which culminates in a less 398 effective priming and differentiation of CD4+ T lymphocytes. We also found a major 399 defect on activation and expansion of B lymphocytes. To begin with, both the total 400 areas of follicles as well as the size of germinal centers were dramatically decreased 401 in spleen of infected IRAK4-/- mice. This was associated with a significant decrease in 402 the number of activated B cells expressing CD86 as well as CD69. TLR activation, 403 either directly on B lymphocytes (20) or follicular dendritic cells (13), is critical for 404


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activity of germinal centers. This defective activation and expansion of B 405 lymphocytes, can be also explained by the impaired CD4+ T cell responses observed 406 in the IRAK4 deficient mice. 407 Importantly, the expression of non-functional IRAK4 in humans results in recurrent 408 life-threatening infections (5, 30). Mutations found in the IRAK4 gene impair the 409 innate and acquired immune responses to microbial TLR ligans and various 410 pathogens of clinical relevance (8, 21, 39). Recent studies have associated the 411 presence of clinical outcomes of congenital and/or acquired toxoplasmosis, in 412 special the development of retinochoroiditis, with genes related to the innate 413 immunity. Thus, we also investigate whether polymorphisms in the IRAK4 gene 414 would associate with the sequelae of congenital toxoplasmosis in humans. In fact, we 415 found evidence of association of two tag SNPs (i.e., rs1461567 and rs4251513) 416 distributed in the IRAK4 gene with brain and ocular alterations in congenital 417 toxoplasmosis. Taken together, the associations of SNPs in TLR9 with 418 retinochoroiditis (37), and in IRAK4 with congenital toxoplasmosis, support the 419 hypothesis that TLRs are critical in mediating resistance to T. gondii infection in 420 humans. 421 In conclusion, innate immunity plays a major role in host resistance to 422 infection with T. gondii in naïve hosts. This is highly relevant to congenital 423 toxoplasmosis, which is mainly transmitted from non-immune pregnant women to 424 the foetus during primary infection. The results presented here indicate that IRAK4 425 plays a critical role in activation and function of splenic dendritic cells and 426 macrophages. As a consequence it is essential for development of acquired 427


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immunity, and thus, an important determinant for host resistance to primary infection 428 with T. gondii and clinical outcome of congenital disease in humans. 429 430


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Acknowledgments 431 This work was supported in part by the National Institute of Health (R01s AI071319-432 01, AI027530-18, AI05042), and the National Institute of Science and Technology for 433 Vaccines (INCTV/CNPq/FAPEMIG). We gratefully acknowledge D. Golenbock for 434 comments and suggestions during the development of this study; the members of 435 the NCCCTS, who performed serologic testing, obtained histories from, examined, 436 and obtained PBMCs from congenitally infected persons; as well as the patients, their 437 families and primary physicians for participating and for permitting us to follow 438 their progress. The NCCCTS physicians and scientists, include K. Boyer, MD, S. 439 Withers, RN, A.G. Noble, MD PhD, C.N. Swisher, MD, P.T. Heydemann, MD, P. Rabiah, 440 MD, D. Burrowes, MD, M. Mets, MD, P. Latkany, MD, W. Mieler, MD, D. McLone, MD, D. 441 Frimm, MD, J.S. Remington, MD, and D. Patel, MD. We also thank the airlines that 442 provided complimentary transportation to Chicago, The Hyatt Hotel Foundation that 443 provided complimentary accommodations for the families during visits in Chicago 444 and support from the Cornwell, Mann, Rooney-Alden, Taub, Mussilami, Engel, Harris, 445 and Pritzker families. 446 447 448 449 450 451 452 453


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Legends to Figures 673 Figure 1. IRAK4 mediates control of parasite replication and influences disease 674 outcome during primary infection with Toxoplasma gondii. Groups of IRAK4-/-, 675 IRAK4+/- and 129/Ola mice were orally infected with 20 cysts of the ME49 strain. (A) 676 Mortality, (B) morbidity, and (C) weight loss (different letters indicate p <0.0001) 677 were assessed for 30 days after infection. Numbers within parenthesis indicate the 678 number of animals used in the experiment shown in panel A and C. In panel B, each 679 dot in one time-point corresponds to one animal. Morbidity was scored as follows: 680 bright and active (0); ruffled coat (1), 10% weight loss (2), hunched, tottering gait, 681 starry stiff coat (3); reluctance to move and 20% weight loss (4) and death (5). Data 682 are representative of two independent experiments that yielded similar results. (D) 683 Tissue cysts are indicated with black arrowhead in IRAK4-/- (left panels), IRAK4+/- 684 (middle panels) and 129 mice (right panels). Black arrows indicate absence or 685 presence of perivascular inflammatory infiltrate in IRAK4-/-, IRAK4+/- and 129 mice, 686 respectively. Asterisk indicates area of moderate necrosis in brain of IRAK4-/- mice. 687 (E) Left graph, cyst counts in histological sections of the brain from IRAK4-/- and 688 IRAK4+/- mice infected with T. gondii. Middle and right graphs show, respectively, the 689 quantification of parasite DNA in the livers and spleens of IRAK-/- and IRAK+/- mice at 690 7 and 9 days after infection. The presented results are an average of eight mice per 691 group and p-values calculated for a 95% confidence interval. The experiments were 692 repeated three times with similar results. 693 Figure 2. Altered structures of follicles and germinal centers in spleen of 694 IRAK4-/- mice infected with T. gondii. (A) Spleens from mice at 7 days post-695 infection were removed and stained with H&E for histological evaluation. Black 696


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arrowhead indicates activated and non-activated germinal centers in spleens from 697 IRAK4-/- (upper panel) and IRAK4+/- (bottom panel) mice, respectively. 698 Quantification of follicles and germinal center areas in spleen from IRAK4-/- and 699 IRAK4+/- mice. (B) Dot plots graphs (frequency) and bars graph (absolute number) 700 show B cells in proliferation (CD19+Ki67+) from IRAK4+/- and IRAK4-/- mice (left 701 panels and right bottom graph). (C) Contour plots, on the left and graphs show, 702 respectively, the frequency and total numbers of activated (i.e., CD86+ or CD69+) B 703 lymphocytes (CD19+). The results of uninfected and infected mice are represented in 704 white and black bars, respectively. The presented results are an average of four mice 705 per group and p-values calculated for a 95% confidence interval. The experiments 706 were repeated three (A) and two (B and C) times with similar results. 707 Figure 3. Impaired cytokine response in IRAK4-/- mice infected with T. gondii. 708 IRAK-/- and IRAK+/- mice were orally infected with ME49 strain of T. gondii. (A) The 709 levels of cytokines in serum were evaluated at 7 days post-infection by ELISA and 710 CBA. p-values were calculated for a 95% confidence interval. (B) Splenocytes from 711 IRAK-/- and IRAK+/- mice were harvested at 7 days post-infection and cultured for 48 712 h without the addition of exogenous stimulus. Cytokine levels were measured in the 713 supernatants by ELISA and CBA. The results of uninfected and infected mice are 714 represented in white and black, respectively. The presented results are an average of 715 eight mice per group and p-values calculated for a 95% confidence interval. The 716 experiment was repeated three times with similar results. 717 Figure 4. IRAK4-dependent IL-12 and TNFα production by macrophages from 718 mice infected with T. gondii. Macrophages of IRAK4+/- and IRAK4-/- mice were 719 analyzed at 7 days after infection with T. gondii. In A, representative dot plots of the 720


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frequency and bar graphs (C – top panels) showing the average number of different 721 subsets of myeloid cells in spleens from IRAK4+/- and IRAK4-/- mice, based on the 722 levels of F4/80, CD11b and GR1 expression in B panel. The percentage of 723 CD11bhiF4/80loGR1hi, CD11bloF4/80hiGR1int and CD11bintF4/80loGR1lo producing IL-724 12 and TNFα are shown in (C) middle and bottom panels, respectively. The results of 725 uninfected and infected mice are represented white and black bars, respectively. The 726 presented results are an average of four mice per group and p-values calculated for a 727 95% confidence interval. The experiment was repeated three times with similar 728 results. 729 Figure 5. IRAK4-dependent production of IL-12 by dendritic cells from mice 730 infected with T. gondii. Dendritic cells (DCs) were analyzed 7 days after infection 731 with T. gondii. (A) Representative dot plots (top panels) show the frequency of DCs 732 (CD11+, MHC-IIhi) in spleens from IRAK4+/- and IRAK4-/- mice. Representative dot 733 plots of frequency of DCs producing IL-12 or TNFα are shown in middle and bottom 734 panels. Bar graphs showing the average number of DCs (CD11+, MHC-IIhi) (top right 735 panel), frequency of DCs producing IL-12 (middle right panel) or TNFα (bottom 736 right panel) in spleens from IRAK4+/- and IRAK4-/- mice. (B) The expression (MFI) 737 of activation the markers CD86 and CD40. (C) Survival curve of the deficient mice 738 treated with or not with recombinant IL-12 during seven days of T. gondii infection. 739 The results of uninfected and infected mice are represented in white and black bars, 740 respectively. The presented results are an average of four mice per group and p-741 values calculated for a 95% confidence interval. The experiment was repeated three 742 (A and B) or twice (C) with similar results. 743


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Figure 6. Impairment of the cell number and IFN-γ-producing T cells in 744 deficient IRAK4 mice. (A) Representative dot plot on the left showing the 745 frequency of CD4+ and CD8+ T cells, within the CD3+ lymphocytes. Bar graphs show 746 the absolute number of CD4+ T as well as CD8+ T lymphocytes from IRAK4+/- and 747 IRAK4-/- mice infected or not with T. gondii. (B) Dot plots illustrating the IFNγ 748 producing-CD4+ T and -CD8+ T lymphocytes stimulated with STAg from IRAK4-/- 749 (lower panel) as compared to IRAK4+/- (upper panel) infected with T. gondii. Bar 750 graphs show the frequency of IFNγ producing-CD4+ T and -CD8+ T lymphocytes 751 infected cultured with STAg. The presented results are an average of four mice per 752 group and p-values calculated for a 95% confidence interval. The experiment was 753 repeated three times (A and B) with similar results. 754 Figure 7. Association of SNPs in the IRAK4 gene with human congenital 755 toxoplasmosis. The diagram shows the positions of genotyped SNPs relative to the 756 intron/exon structure of the gene. Lower diagram shows the LD plots generated in 757 Haploview using IRAK4 gene data from our North American patient cohort. LD 758 values (D′×100) between markers are indicated at the intercept of the 2 markers on 759 the matrix. Where there is no value, D′=1 (i.e. 100). In outlining gene SNP’s 760 association with susceptibility to congenital toxoplasmosis, D' values between loci 761 were calculated and displayed using Haploview. When there is high confidence in the 762 value of D' (LOD score >2) shades of pink red and bright red are used. If the 763 confidence is lower (LOD score <2), blue shading (D'=1) and no shading (D'<1) are 764 used. 765 766


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impaired innate immunity in irak4 deficient mice leads to defective

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