IRAK‐M regulates the inhibition of TLR‐mediated macrophage immune response during late in vitro Leishmania donovani infection

Abstract

Intramacrophage protozoan parasite Leishmania donovani, causative agent of visceral leishmaniasis, escapes Toll-like receptor (TLR) dependent early host immune response by inducing the deubiquitinating enzyme A20, which is sustained up to 6 h postinfection only. Therefore, Leishmania must apply other means to deactivate late host responses. Here, we elucidated the role of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR signaling, in downregulating macrophage proinflammatory response during late hours of in vitro infection. Our data reveal a sharp decline in IRAK1 and IRAK4 phosphorylation at 24 h postinfection along with markedly reduced association of IRAK1-TNF receptor associated factor 6, which is mandatory for TLR activation. In contrast, IRAK-M was induced after A20 levels decreased and reached a maximum at 24 h postinfection. IRAK-M induction coincided with increased stimulation of TGF-β, a hallmark cytokine of visceral infection. TGF-β-dependent signaling-mediated induction of SMAD family of proteins, 2, 3, and 4 plays important roles in transcriptional upregulation of IRAK-M. In infected macrophages, siRNA-mediated silencing of IRAK-M displayed enhanced IRAK1 and IRAK4 phosphorylation with a concomitant increase in downstream NF-κB activity and reduced parasite survival. Taken together, the results suggest that IRAK-M may be targeted by L. donovani to inhibit TLR-mediated proinflammatory response late during in vitro infection.Keywords: Host defense r IRAK-M r Leishmania r Macrophage r Toll-like receptors Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionVisceral leishmaniasis caused by the protozoan parasite Leishmania donovani is associated with immunological dysfunctions of macrophages [1]. In spite of multiple intracellular signal transduction pathways stringently regulating macrophage effector functions, the parasite gets access into the host macrophages and Correspondence: Dr. Pijush K. Das e-mail: pijushdas@iicb.res.in develops several strategies to secure its own survival and replication [2]. The innate immune response against L. donovani infection begins with the recognition of molecular structures related to this pathogen by Toll-like receptors (TLRs) [3]. Upon activation, TLRs associate with the myeloid differentiation factor 88 (MyD88), which further recruits IL-1 receptor associated kinase (IRAK) proteins and TNF receptor associated factor 6 (TRAF6) [4]. This is followed by a series of events culminating in the degradation of IκB, thereby allowing NF-κB to be translocated to the nucleus and to activate the transcription of proinflammatory cytokines such as . Although inflammatory response is C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 2788 Supriya Srivastav et al. Eur. J. Immunol. 2015. 45: 2787-2797 critical to control the growth of pathogenic organisms, uncontrolled stimulation of TLRs can lead to disproportionate inflammation. Therefore, TLR signaling is tightly re...

Leishmaniatargets the macrophage epigenome and dampens the NF-κB/NLRP3-mediated inflammatory response

“…While the underlying parasite effector mechanisms causing host cell immune suppression remain elusive, our study draws a detailed and complex picture of their impact on the host cell phenotype. We reveal a long-lasting (over three weeks) and global inhibition of the NF-B-mediated pro-inflammatory response on the transcriptomics level, which extends previous findings on specific inhibition of this pathway early during infection involving cleavage of individual NF-B members (Abu-Dayyeh et al, 2010;Calegari-Silva et al, 2009;Cameron et al, 2004;Gregory et al, 2008), the formation of Crel/P50 or P50/P50 dimers (Calegari-Silva et al, 2009;Guizani-Tabbane et al, 2004), the specific increase of key inhibitors of TLR-signaling such as TNFAIP3 or IRAKm (Srivastav et al, 2012;Srivastav et al, 2015), or the inhibition of TRAF3 degradation (Gupta et al, 2017). Thus, L. amazonensis not only avoids inflammasone activation early during infection allowing for establishment of macrophage infection, but reprograms its host cell into a safe haven permissive for long-term persistent infection and intracellular parasite growth.…”

Recent advances in understanding Leishmania donovani infection: The importance of diverse host regulatory pathways

“…We showed that IRAK‐M is exploited by L. donovani to exercise a sustained repression on TLR‐mediated pro‐inflammatory response during established infection . In infected macrophages, siRNA mediated silencing of IRAK‐M displayed enhanced IRAK1 and IRAK4 phosphorylation, which is mandatory for TLR activation with a concomitant increase in downstream NF‐κB activity and reduced parasite burden . IRAK‐1 inactivation during Leishmania infection has been reported in another study where SHP‐1 was exploited by Leishmania to bind to IRAK‐1 and for complete suppression of its intrinsic kinase activity .…”

“…Moreover, enhanced innate immunity was found in IRAK‐M knockout mice as revealed by increased inflammatory responses against bacterial infection. We showed that IRAK‐M is exploited by L. donovani to exercise a sustained repression on TLR‐mediated pro‐inflammatory response during established infection . In infected macrophages, siRNA mediated silencing of IRAK‐M displayed enhanced IRAK1 and IRAK4 phosphorylation, which is mandatory for TLR activation with a concomitant increase in downstream NF‐κB activity and reduced parasite burden .…”

Neutrophil elastase promotes Leishmania donovani infection via interferon‐β

“…Even though we found that macrophage infection in vitro by L. donovani requires TLR4 for parasite growth, it is likely that TLR4 present in additional cell types besides macrophages plays a role during experimental infection, contributing to parasite control. Using a monocytic cell line RAW or bone marrow–derived macrophages, Srivastav et al (36) reported that L. donovani AG83 deactivates the TNF receptor-associated factor (TRAF)6–interleukin-1 receptor-associated kinase (IRAK)1 (TRAF6-IRAK1) complex during long-term infection and sustains elevated levels of the TLR-negative regulator IRAK-M. Even though L. donovani was found to enhance the stability of the IRAK1-myeloid differentiation primary response 88 (MyD88) complex, thus potentially deactivating TLR-mediated responses, the consequences of the direct activation of TLR2 or TLR4 in infected macrophages were not directly addressed.…”