Group 1 ILCs: Heterogeneity, plasticity, and transcriptional regulation.

Group 1 innate lymphoid cells (ILCs), comprising ILC1s and natural killer cells (NK cells), belong to a large family of developmentally related innate lymphoid cells that lack rearranged antigen-specific receptors. NK cells and ILC1s both require the transcription factor T-bet for lineage commitment but additionally rely on Eomes and Hobit, respectively, for their development and effector maturation programs. Both ILC1s and NK cells are essential for rapid responses against infections and mediate cancer immunity through production of effector cytokines and cytotoxicity mediators. ILC1s are enriched in tissues and hence generally considered tissue resident cells whereas NK cells are often considered circulatory. Despite being deemed different cell types, ILC1s and NK cells share many common features both phenotypically and functionally. Recent studies employing single cell RNA sequencing (scRNA-seq) technology have exposed previously unappreciated heterogeneity in group 1 ILCs and further broaden our understanding of these cells. Findings from these studies imply that ILC1s in different tissues and organs share a common signature but exhibit some unique characteristics, possibly stemming from tissue imprinting. Also, data from recent fate mapping studies employing Hobit, RORγt, and polychromic reporter mice have greatly advanced our understanding of the developmental and effector maturation programs of these cells. In this review, we aim to outline the fundamental traits of mouse group 1 ILCs and explore recent discoveries related to their developmental programs, phenotypic heterogeneity, plasticity, and transcriptional regulation.

Inositol phosphatase INPP4B sustains ILC1s and intratumoral NK cells through an AKT-driven pathway.

Innate lymphoid cells (ILCs) are a heterogeneous population of lymphocytes that coordinate early immune responses and maintain tissue homeostasis. Type 1 innate immune responses are mediated by natural killer (NK) cells and group 1 ILCs (ILC1s). Despite their shared features, NK cells and ILC1s display profound differences among various tissue microenvironments. Here, we identify the inositol polyphosphatase INPP4B as a hallmark feature of tissue-resident ILC1s and intratumoral NK cells using an scRNA-seq atlas of tissue-associated and circulating NK/ILC1s. Conditional deletion of Inpp4b in ILC1s and NK cells reveals that it is necessary for the homeostasis of tissue-resident ILC1s but not circulating NK cells at steady-state. Inpp4b-deficient cells display increased rates of apoptosis and reduced activation of the prosurvival molecule AKT. Furthermore, expression of Inpp4b by NK/ILC1s is necessary for their presence in the intratumoral environment, and lack of Inpp4b impairs antitumor immunity. These findings highlight INPP4B as a novel regulator of tissue residency and antitumor function in ILC1s and NK cells.

The transcription factor Aiolos restrains the activation of intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) exhibit prompt innate-like responses to microenvironmental cues and require strict control of effector functions. Here we showed that Aiolos, an Ikaros zinc-finger family member encoded by Ikzf3, acted as a regulator of IEL activation. Ikzf3-/- CD8αα+ IELs had elevated expression of NK receptors, cytotoxic enzymes, cytokines and chemokines. Single-cell RNA sequencing of Ikzf3-/- and Ikzf3+/+ IELs showed an amplified effector machinery in Ikzf3-/- CD8αα+ IELs compared to Ikzf3+/+ counterparts. Ikzf3-/- CD8αα+ IELs had increased responsiveness to interleukin-15, which explained a substantial part, but not all, of the observed phenotypes. Aiolos binding sites were close to those for the transcription factors STAT5 and RUNX, which promote interleukin-15 signaling and cytolytic programs, and Ikzf3 deficiency partially increased chromatin accessibility and histone acetylation in these regions. Ikzf3 deficiency in mice enhanced susceptibility to colitis, underscoring the relevance of Aiolos in regulating the effector function in IELs.

SHIP1 inhibition via 3-alpha-amino-cholestane enhances protection against Leishmania infection.

Leishmania major and L. donovani cause cutaneous leishmaniasis and visceral leishmaniasis, respectively. Available chemotherapies suffer from toxicity, drug-resistance or high cost of production prompting the need for the discovery of new anti-leishmanials. Here, we test a novel aminosteriodal compound- 3-alpha-amino-cholestane [3AC] - that shows selective inhibition of SHIP1, an inositol-5'-phosphate-specific phosphatase with potent effects on the immune system. We report that 3AC-sensitive SHIP1 expression increases in Leishmania-infected macrophages. Treatment of BALB/c mice, a Leishmania-susceptible host, with 3AC increased anti-leishmanial, but reduced pro-leishmanial, cytokines' production and reduced the parasite load in both L. major and L. donovani infections. These findings implicate SHIPi as a potential novel immunostimulant with anti-leishmanial function.

Obesity control by SHIP inhibition requires pan-paralog inhibition and an intact eosinophil compartment.

Here we extend the understanding of how chemical inhibition of SHIP paralogs controls obesity. We compare different classes of SHIP inhibitors and find that selective inhibitors of SHIP1 or SHIP2 are unable to prevent weight gain and body fat accumulation during increased caloric intake. Surprisingly, only pan-SHIP1/2 inhibitors (pan-SHIPi) prevent diet-induced obesity. We confirm that pan-SHIPi is essential by showing that dual treatment with SHIP1 and SHIP2 selective inhibitors reduced adiposity during excess caloric intake. Consistent with this, genetic inactivation of both SHIP paralogs in eosinophils or myeloid cells also reduces obesity and adiposity. In fact, pan-SHIPi requires an eosinophil compartment to prevent diet-induced adiposity, demonstrating that pan-SHIPi acts via an immune mechanism. We also find that pan-SHIPi increases ILC2 cell function in aged, obese mice to reduce their obesity. Finally, we show that pan-SHIPi also reduces hyperglycemia, but not via eosinophils, indicating a separate mechanism for glucose control.

Repression of the aryl-hydrocarbon receptor prevents oxidative stress and ferroptosis of intestinal intraepithelial lymphocytes.

The aryl-hydrocarbon receptor (AHR) is a ligand-activated transcription factor that buoys intestinal immune responses. AHR induces its own negative regulator, the AHR repressor (AHRR). Here, we show that AHRR is vital to sustaining intestinal intraepithelial lymphocytes (IELs). AHRR deficiency reduced IEL representation in a cell-intrinsic fashion. Single-cell RNA sequencing revealed an oxidative stress profile in Ahrr-/- IELs. AHRR deficiency unleashed AHR-induced expression of CYP1A1, a monooxygenase that generates reactive oxygen species, increasing redox imbalance, lipid peroxidation, and ferroptosis in Ahrr-/- IELs. Dietary supplementation with selenium or vitamin E to restore redox homeostasis rescued Ahrr-/- IELs. Loss of IELs in Ahrr-/- mice caused susceptibility to Clostridium difficile infection and dextran sodium-sulfate-induced colitis. Inflamed tissue of inflammatory bowel disease patients showed reduced Ahrr expression that may contribute to disease. We conclude that AHR signaling must be tightly regulated to prevent oxidative stress and ferroptosis of IELs and to preserve intestinal immune responses.

SLC7A8 is a key amino acids supplier for the metabolic programs that sustain homeostasis and activation of type 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2) are innate counterparts of T helper 2 (Th2) cells that maintain tissue homeostasis and respond to injuries through rapid interleukin (IL)-5 and IL-13 secretion. ILC2s depend on availability of arginine and branched-chain amino acids for sustaining cellular fitness, proliferation, and cytokine secretion in both steady state and upon activation. However, the contribution of amino acid transporters to ILC2 functions is not known. Here, we found that ILC2s selectively express Slc7a8, encoding a transporter for arginine and large amino acids. Slc7a8 was expressed in ILC2s in a tissue-specific manner in steady state and was further increased upon activation. Genetic ablation of Slc7a8 in lymphocytes reduced the frequency of ILC2s, suppressed IL-5 and IL-13 production upon stimulation, and impaired type 2 immune responses to helminth infection. Consistent with this, Slc7a8-deficient ILC2s also failed to induce cytokine production and recruit eosinophils in a model of allergic lung inflammation. Mechanistically, reduced amino acid availability due to Slc7a8 deficiency led to compromised mitochondrial oxidative phosphorylation, as well as impaired activation of mammalian target of rapamycin and c-Myc signaling pathways. These findings identify Slc7a8 as a key supplier of amino acids for the metabolic programs underpinning fitness and activation of ILC2s.

TREM2 drives microglia response to amyloid-ß via SYK-dependent and -independent pathways.

Genetic studies have highlighted microglia as pivotal in orchestrating Alzheimer's disease (AD). Microglia that adhere to Aß plaques acquire a transcriptional signature, "disease-associated microglia" (DAM), which largely emanates from the TREM2-DAP12 receptor complex that transmits intracellular signals through the protein tyrosine kinase SYK. The human TREM2R47H variant associated with high AD risk fails to activate microglia via SYK. We found that SYK-deficient microglia cannot encase Aß plaques, accelerating brain pathology and behavioral deficits. SYK deficiency impaired the PI3K-AKT-GSK-3ß-mTOR pathway, incapacitating anabolic support required for attaining the DAM profile. However, SYK-deficient microglia proliferated and advanced to an Apoe-expressing prodromal stage of DAM; this pathway relied on the adapter DAP10, which also binds TREM2. Thus, microglial responses to Aß involve non-redundant SYK- and DAP10-pathways. Systemic administration of an antibody against CLEC7A, a receptor that directly activates SYK, rescued microglia activation in mice expressing the TREM2R47H allele, unveiling new options for AD immunotherapy.

LRBA Deficiency Can Lead to Lethal Colitis That Is Diminished by SHIP1 Agonism.

Humans homozygous for inactivating LRBA (lipopolysaccharide (LPS)-responsive beige-like anchor) mutations or with compound heterozygous mutations exhibit a spectrum of immune-related pathologies including inflammatory bowel disease (IBD). The cause of this pathology remains undefined. Here we show that disruption of the colon epithelial barrier in LRBA-deficient mice by dextran sulfate sodium (DSS) consumption leads to severe and uniformly lethal colitis. Analysis of bone marrow (BM) chimeras showed that susceptibility to lethal colitis is primarily due to LRBA deficiency in the immune compartment and not the gut epithelium. Further dissection of the immune defect in LRBA-deficient hosts showed that LRBA is essential for the expression of CTLA4 by Treg cells and IL22 and IL17 expression by ILC3 cells in the large intestine when the gut epithelium is compromised by DSS. We further show that SHIP1 agonism partially abrogates the severity and lethality of DSS-mediated colitis. Our findings indicate that enteropathy induced by LRBA deficiency has multiple causes and that SHIP1 agonism can partially abrogate the inflammatory milieu in the gut of LRBA-deficient hosts.

Multi-tissue single-cell analysis deconstructs the complex programs of mouse natural killer and type 1 innate lymphoid cells in tissues and circulation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are heterogenous innate lymphocytes broadly defined in mice as Lin-NK1.1+NKp46+ cells that express the transcription factor T-BET and produce interferon-γ. The ILC1 definition primarily stems from studies on liver and small intestinal populations. However, NK1.1+NKp46+ cells in the salivary glands, uterus, adipose, and other tissues exhibit nonuniform programs that differ from those of liver or intestinal ILC1s or NK cells. Here, we performed single-cell RNA sequencing on murine NK1.1+NKp46+ cells from blood, spleen, various tissues, and solid tumors. We identified gene expression programs of tissue-specific ILC1s, tissue-specific NK cells, and non-tissue-specific populations in blood, spleen, and other tissues largely corresponding to circulating cells. Moreover, we found that circulating NK cell programs were reshaped in tumor-bearing mice. Core programs of circulating and tumor NK cells paralleled conserved human NK cells signatures, advancing our understanding of the human NK-ILC1 spectrum.

Differential usage of transcriptional repressor Zeb2 enhancers distinguishes adult and embryonic hematopoiesis.

The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural, and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400-basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400-bp region (Zeb2Δ-165mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC)-derived lineages. Zeb2Δ-165 mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2Δ-165 mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at +164-kb that was selectively active in embryonically derived lineages, but not HSC-derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the -165-kb Zeb2 enhancer.

LmjF.36.3850, a novel hypothetical Leishmania major protein, contributes to the infection.

Leishmania is a protozoan parasite that resides in mammalian macrophages and inflicts the disease known as leishmaniasis. Although prevalent in 88 countries, an anti-leishmanial vaccine remains elusive. While comparing the virulent and avirulent L. major transcriptomes by microarray, PCR and functional analyses for identifying a novel virulence-associated gene, we identified LmjF.36.3850, a hypothetical protein significantly less expressed in the avirulent parasite and without any known function. Motif search revealed that LmjF.36.3850 protein shared phosphorylation sites and other structural features with sucrose non-fermenting protein (Snf7) that shuttles virulence factors. LmjF.36.3850 was predicted to bind diacylglycerol (DAG) with energy value similar to PKCα and PKCß, to which DAG is a cofactor. Indeed, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue, enhanced the phosphorylation of PKCα and PKCßI. We cloned LmjF.36.3850 gene in a mammalian expression vector and primed susceptible BALB/c mice followed by challenge infection. We observed a higher parasite load, comparable antibody response and higher anti-inflammatory cytokines such as IL-4 and IL-10, while expression of major anti-leishmanial cytokine, IFN-γ, remained unchanged in LmjF.36.3850-vaccinated mice. CSA restimulated LN cells from vaccinated mice after challenge infection secreted comparable IL-4 and IL-10 but reduced IFN-γ, as compared to controls. These observations suggest a skewed Th2 response, diminished IFN-γ secreting Th1-TEM cells and increased central and effector memory subtype of Th2, Th17 and Treg cells in the vaccinated mice. These data indicate that LmjF.36.3850 is a plausible virulence factor that enhances disease-promoting response, possibly by interfering with PKC activation and by eliciting disease-promoting T cells.

SHIP1 Deficiency in Inflammatory Bowel Disease Is Associated With Severe Crohn's Disease and Peripheral T Cell Reduction.

In our previous study, we observed a severe reduction in the Src homology 2-containing-inositol-phosphatase-1 (SHIP1) protein in a subpopulation of subjects from a small adult Crohn's Disease (CD) cohort. This pilot study had been undertaken since we had previously demonstrated that engineered deficiency of SHIP1 in mice results in a spontaneous and severe CD-like ileitis. Here, we extend our analysis of SHIP1 expression in peripheral blood mononuclear cells in a second much larger adult Inflammatory Bowel Disease (IBD) cohort, comprised of both CD and Ulcerative Colitis patients, to assess contribution of SHIP1 to the pathogenesis of human IBD. SHIP1 protein and mRNA levels were evaluated from blood samples obtained from IBD subjects seen at UCSF/SFVA, and compared to healthy control samples. Western blot analyses revealed that ~15% of the IBD subjects are severely SHIP1-deficient, with less than 10% of normal SHIP1 protein present in PBMC. Further analyses by flow cytometry and sequencing were performed on secondary samples obtained from the same subjects. Pan-hematolymphoid SHIP1 deficiency was a stable phenotype and was not due to coding changes in the INPP5D gene. A very strong association between SHIP1 deficiency and the presence of a novel SHIP1:ATG16L1 fusion transcript was seen. Similar to SHIP1-deficient mice, SHIP1-deficient subjects had reduced numbers of circulating CD4+ T cell numbers. Finally, SHIP1-deficient subjects with CD had a history of severe disease requiring multiple surgeries. These studies reveal that the SHIP1 protein is crucial for normal T cell homeostasis in both humans and mice, and that it is also a potential therapeutic and/or diagnostic target in human IBD.

Dual enhancement of T and NK cell function by pulsatile inhibition of SHIP1 improves antitumor immunity and survival.

The success of immunotherapy in some cancer patients has revealed the profound capacity for cytotoxic lymphocytes to eradicate malignancies. Various immunotherapies work by blocking key checkpoint proteins that suppress immune cell activity. The phosphatase SHIP1 (SH2-containing inositol polyphosphate 5-phosphatase) limits signaling from receptors that activate natural killer (NK) cells and T cells. However, unexpectedly, genetic ablation studies have shown that the effector functions of SHIP1-deficient NK and T cells are compromised in vivo. Because chronic activation of immune cells renders them less responsive to activating signals (a host mechanism to avoid autoimmunity), we hypothesized that the failure of SHIP1 inhibition to induce antitumor immunity in those studies was caused by the permanence of genetic ablation. Accordingly, we found that reversible and pulsatile inhibition of SHIP1 with 3-α-aminocholestane (3AC; "SHIPi") increased the antitumor response of NK and CD8+ T cells in vitro and in vivo. Transient SHIP1 inhibition in mouse models of lymphoma and colon cancer improved the median and long-term tumor-free survival rates. Adoptive transfer assays showed evidence of immunological memory to the tumor in hematolymphoid cells from SHIPi-treated, long-term surviving mice. The findings suggest that a pulsatile regimen of SHIP1 inhibition might be an effective immunotherapy in some cancer patients.

LRBA is Essential for Allogeneic Responses in Bone Marrow Transplantation.

The PH-BEACH-WD40 (PBW) protein family members play a role in coordinating receptor signaling and intracellular vesicle trafficking. LPS-Responsive-Beige-like Anchor (LRBA) is a PBW protein whose immune function remains elusive. Here we show that LRBA-null mice are viable, but exhibit compromised rejection of allogeneic, xenogeneic and missing self bone-marrow grafts. Further, we demonstrate that LRBA-null Natural Killer (NK) cells exhibit impaired signaling by the key NK activating receptors, NKp46 and NKG2D. However, induction of IFN-γ by cytokines remains intact, indicating LRBA selectively facilitates signals by receptors for ligands expressed on the surface of NK targets. Surprisingly, LRBA limits immunoregulatory cell numbers in tissues where GvHD is primed or initiated, and consistent with this LRBA-null mice also demonstrate resistance to lethal GvHD. These findings demonstrate that LRBA is redundant for host longevity while being essential for both host and donor-mediated immune responses and thus represents a unique and novel molecular target in transplant immunology.

A small-molecule inhibitor of SHIP1 reverses age- and diet-associated obesity and metabolic syndrome.

Low-grade chronic inflammation is a key etiological phenomenon responsible for the initiation and perpetuation of obesity and diabetes. Novel therapeutic approaches that can specifically target inflammatory pathways are needed to avert this looming epidemic of metabolic disorders. Genetic and chemical inhibition of SH2-containing inositol 5' phosphatase 1 (SHIP1) has been associated with systemic expansion of immunoregulatory cells that promote a lean-body state; however, SHIP1 function in immunometabolism has never been assessed. This led us to investigate the role of SHIP1 in metabolic disorders during excess caloric intake in mice. Using a small-molecule inhibitor of SHIP1 (SHIPi), here we show that SHIPi treatment in mice significantly reduces body weight and fat content, improves control of blood glucose and insulin sensitivity, and increases energy expenditure, despite continued consumption of a high-fat diet. Additionally, SHIPi reduces age-associated fat in mice. We found that SHIPi treatment reverses diet-associated obesity by attenuating inflammation in the visceral adipose tissue (VAT). SHIPi treatment increases IL-4-producing eosinophils in VAT and consequently increases both alternatively activated macrophages and myeloid-derived suppressor cells. In addition, SHIPi decreases the number of IFN-γ-producing T cells and NK cells in VAT. Thus, SHIPi represents an approach that permits control of obesity and diet-induced metabolic syndrome without apparent toxicity.

Role of inositol poly-phosphatases and their targets in T cell biology.

T lymphocytes play a critical role in host defense in all anatomical sites including mucosal surfaces. This not only includes the effector arm of the immune system, but also regulation of immune responses in order to prevent autoimmunity. Genetic targeting of PI3K isoforms suggests that generation of PI(3,4,5)P3 by PI3K plays a critical role in promoting effector T cell responses. Consequently, the 5'- and 3'-inositol poly-phosphatases SHIP1, SHIP2, and phosphatase and tensin homolog capable of targeting PI(3,4,5)P3 are potential genetic determinants of T cell effector functions in vivo. In addition, the 5'-inositol poly-phosphatases SHIP1 and 2 can shunt PI(3,4,5)P3 to the rare but potent signaling phosphoinositide species PI(3,4)P2 and thus these SHIP1/2, and the INPP4A/B enzymes that deplete PI(3,4)P2 may have precise roles in T cell biology to amplify or inhibit effectors of PI3K signaling that are selectively recruited to and activated by PI(3,4)P2. Here we summarize recent genetic and chemical evidence that indicates the inositol poly-phosphatases have important roles in both the effector and regulatory functions of the T cell compartment. In addition, we will discuss future genetic studies that might be undertaken to further elaborate the role of these enzymes in T cell biology as well as potential pharmaceutical manipulation of these enzymes for therapeutic purposes in disease settings where T cell function is a key in vivo target.

Modeling and experimental analyses reveals signaling plasticity in a bi-modular assembly of CD40 receptor activated kinases.

Depending on the strength of signal dose, CD40 receptor (CD40) controls ERK-1/2 and p38MAPK activation. At low signal dose, ERK-1/2 is maximally phosphorylated but p38MAPK is minimally phosphorylated; as the signal dose increases, ERK-1/2 phosphorylation is reduced whereas p38MAPK phosphorylation is reciprocally enhanced. The mechanism of reciprocal activation of these two MAPKs remains un-elucidated. Here, our computational model, coupled to experimental perturbations, shows that the observed reciprocity is a system-level behavior of an assembly of kinases arranged in two modules. Experimental perturbations with kinase inhibitors suggest that a minimum of two trans-modular negative feedback loops are required to reproduce the experimentally observed reciprocity. The bi-modular architecture of the signaling pathways endows the system with an inherent plasticity which is further expressed in the skewing of the CD40-induced productions of IL-10 and IL-12, the respective anti-inflammatory and pro-inflammatory cytokines. Targeting the plasticity of CD40 signaling significantly reduces Leishmania major infection in a susceptible mouse strain. Thus, for the first time, using CD40 signaling as a model, we show how a bi-modular assembly of kinases imposes reciprocity to a receptor signaling. The findings unravel that the signalling plasticity is inherent to a reciprocal system and that the principle can be used for designing a therapy.

Reciprocal regulation of protein kinase C isoforms results in differential cellular responsiveness.

Immunological homeostasis is often maintained by counteractive functions of two different cell types or two different receptors signaling through different intermediates in the same cell. One of these signaling intermediates is protein kinase C (PKC). Ten differentially regulated PKC isoforms are integral to receptor-triggered responses in different cells. So far, eight PKC isoforms are reported to be expressed in macrophages. Whether a single receptor differentially uses PKC isoforms to regulate counteractive effector functions has never been addressed. As CD40 is the only receptor characterized to trigger counteractive functions, we examined the relative role of PKC isoforms in the CD40-induced macrophage functions. We report that in BALB/c mouse macrophages, higher doses of CD40 stimulation induce optimum phosphorylation and translocation of PKCα, ßI, ßII, and ε whereas lower doses of CD40 stimulation activates PKCδ, ζ, and λ. Infection of macrophages with the protozoan parasite Leishmania major impairs PKCα, ßI, ßII, and ε isoforms but enhances PKCδ, ζ, and λ isoforms, suggesting a reciprocity among these PKC isoforms. Indeed, PKCα, ßI, ßII, and ε isoforms mediate CD40-induced p38MAPK phosphorylation, IL-12 expression, and Leishmania killing; PKCδ and ζ/λ mediate ERK1/2 phosphorylation, IL-10 production, and parasite growth. Treatment of the susceptible BALB/c mice with the lentivirally expressed PKCδ- or ζ-specific short hairpin RNA significantly reduces the infection and reinstates host-protective IFN-γ-dominated T cell response, defining the differential roles for PKC isoforms in immune homeostasis and novel PKC-targeted immunotherapeutic and parasite-derived immune evasion strategies.

CD40-modulated dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-3 reciprocally regulate Leishmania major infection.

The macrophage-expressed CD40 regulates immune responses to Leishmania major infection by reciprocal signaling through p38 MAPK and ERK1/2. CD40-induced IL-10 or IL-12 plays crucial roles in the promotion or protection from L. major infection, respectively. Because p38 MAPK and ERK1/2 are dephosphorylated by dual-specificity MAPK phosphatases (MKPs), we tested the role of CD40 in the regulation of MKPs in L. major infection. MKP-1 expression and activity increased whereas MKP-3 expression and activity decreased in virulent L. major-infected macrophages. CD40 differentially regulated the expression and activity of MKP-1 and MKP-3, which, in turn, reciprocally regulated CD40-induced p38 MAPK and ERK1/2 phosphorylation and effector functions in macrophages. Triptolide, an inhibitor of MKP-1 expression, and lentivirally expressed MKP-1 short hairpin RNA enhanced CD40-induced anti-leishmanial functions and significantly protected susceptible BALB/c mice from L. major infection. Similarly, lentivirally overexpressed MKP-3 significantly reduced disease progression and parasite burden in susceptible BALB/c mice. Thus, to our knowledge, our data show for the first time that CD40 reciprocally regulates MKP-1 and MKP-3 expression and activity while the MKPs contribute to the reciprocal CD40 signaling-regulated anti-leishmanial functions. The findings reveal a novel parasite-devised immune evasion strategy and an effective target to redirect CD40-regulated immune responses.