Transcriptional factor Kruppel-like factor 2 is found to be indispensible in numerous pathways that regulate lung and skeletal development, T cell survival and migration, and vascular integrity (34, 35, 36, 44, 67). Besides these established ones, its roles in inflammation are raised to focus recently (27, 37, 43, 65, 66). Overexpression of KLF2 was reported to present an anti-inflammatory phenotype in cells by downregulating some proinflammatory cytokines and chemokines production. Cells which were investigated include endothelial cells, T cells, monocytes, and macrophages, two types of which belong to myeloid lineage. Myeloid cells, as the first line defense against pathogen, are critical in innate immunity against infection. Therefore, the functionality of KLF2 in myeloid cells suggested that some pathogens may utilize this pathway to regulate immune response to its advantage for survival. But so far, no studies have been conducted to identify KLF2's function in myeloid innate immunity upon infection.
Histoplasma capsulatum (Hc), a dimorphic fungus, is a facultative intracellular pathogen that resides in macrophages. Microconidia or mycelial fragments is able to convert to yeast phase at 37°C after inhalation, and subsequently causes restricted or disseminated infection depending on host immunity (68-70). In immunecompetent hosts, natural resolution needs the cooperation between T cells and macrophages. The latter is a key player in innate immunity that restrains Hc intracellular growth by activating cytokines and chemokines production since macrophages are not capable of killing the fungus efficiently though oxidative burst, apoptosis, digestion or limiting iron acquisition (71-75).
In addition to the characteristics of Hc, which makes it an ideal model for study the inflammatory response exerted by macrophages dependent or independent of KLF2, alveolar macrophages (AMs) have some indirect evidences that suggest the involvement of KLF2 with macrophage activation and polarization through peroxisome proliferator activated receptors gamma (PPAR-γ) (1, 8, 9, 19, 45-50). Hence, KLF2 may also have the ability to discriminate between classically activated or alternatively activated macrophages, which subsequently cause discrepancies in inflammatory response. Therefore, it is very compelling for us to study if KLF2 is crucial in macrophage responsiveness and function against a macrophage-resident pathogen Hc.
The hypotheses for these studies include: 1) KLF2 is essential for macrophage polarization, deciding if pathogen can be effectively cleared; 2) KLF2 expression regulate proinflammatory cytokine production in macrophage, thereby modulating the level of immune response towards Hc; and 3) KLF2 expression regulates chemokine production in myeloid cells, which subsequently adjust cell recruitment to infection site.
The specific aims are:
Specific Aim 1. To determine how KLF2 deficiency in macrophages influences immune response to Hc in vitro. Specific projects include: 1) To study if KLF2 deficiency affect macrophage phenotype, including M1/M2 ratio and polarized macrophage marker expression; 2) To determine if Hc infection would change macrophage polarization; 3) To determine if Hc infection affect proinflammatory or anti-inflammatory cytokine production in KLF2 knock out macrophage.
Specific Aim 2. To identify how KLF2 deficiency in myeloid cells influences immune response of Hc in vivo by further studying the phenotype of KLF2 KO mice, including: 1) To study if lethal Hc challenge leads to faster death in KO mice due to lethal fungal load in lung; 2) To study that under subletal Hc challenge, if cytokine production, cell distribution and resolution of infection in KO differs from WT; 3) To determine if production of cytokines and chemokines is different between WT and KO mice, and if it is organ specific by corresponding their levels in blood to those in lung.
Specific Aim 3. To understand the mechanism of how KLF2 plays a role in resolving Hc infection in macrophage. Specific projects include: 1) To study if Th1/Th2 type cytokines can successfully polarize inactivated macrophages when KLF2 is deficient; 2) To study if the level of Th1 cytokine receptor (IFN-γR) or Th2 cytokine receptor (IL-4Rα, PPAR-γ) is different in KO versus WT; 2) To explore other possible mechanisms including iron acquisition, phagocytosis, alternative reactive oxygen species production, apoptosis.
B. RESEARCH STRATEGY
1. Significance. Histoplasma Capsulatum (Hc), as one of the important intracellular pathogens spanning from bacteria, fungi, to parasites, survives and replicates in macrophage in unique ways as compared with extracellular pathogens. Previous researches have shown that most intracellular pathogens escape the killing mechanisms by changing the microenvironment of cell, such as inhibiting phagolysosome fusion, elevating pH within phagolysosomes, and tolerating respiratory burst. Therefore, macrophages as the ultimate effector cells to resolve infection are instrumental for host survival.
Some novel ideas which were raised from another intracellular pathogen mycobacterium tuberculosis (MTb) provided us insights into how macrophages take effect in resolving infection. Alveolar macrophages are prototypic alternatively activated macrophage (AAMs) dominant upon infection (82, 83). Therefore, AAMs governs the clearance of less invasive extracellular pathogens in immunosuppressive manner, and they are inefficient in clearing intracellular pathogens. A critical switching point that change the dominant phenotype of macrophages is controlled by a transcription factor PPAR-γ, priming AAMs towards classically activated macrophages (CAMs), and hence increasing proinflammatory cytokines and chemokines secretion which attract plethora of inflammatory cells to the infection sites (1, 19, 81). Given the fact that Hc also triggers Th1-like response, and the growth restriction by macrophages requires IFN-γ and granulocyte macrophage colony stimulating factor (GM-CSF) which are essential in CAMs activation (84, 85), the switching point for Hc might also exist but happen at much earlier time as compared to MTb.
Our preliminary studies discovered that KLF2 expression was progressively downregulated in Hc infected lung (predominantly in macrophages) for 7 days, and was gradually restored afterwards. This is emerging evidence that KLF2 is regulated throughout the course of infection. Also, KLF2 deficient macrophages enhanced the expression of PPAR-γ, and presented a dominant AAMs phenotype in peritoneal macrophages (PMs) and bone marrow derived macrophages (BMDMs). Hence, the combat between host and Hc appears to center on KLF2 to discriminate inflammatory response, possibly dependent on PPAR-γ. In addition, KLF2 overexpression could downregulate PPAR-γ expression by inhibiting PPAR-γ promoter activity, suggesting KLF2 may be upstream of PPAR-γ (8, 9). Collectively, these evidences are highly indicative of KLF2's modulatory role in macrophage biology and function. By studying the functionality of KLF2 in myeloid immunity will reveal a new component of equilibrium in inflammatory response.
2. Innovation. This proposal will determine the biological and functional role of KLF2 in the context of Hc infection. As a newly identified transcription factor, KLF2 as a regulator of balancing pro- or anti-inflammatory is a brand new perspective. Upon this framework, KLF2 as an upstream signal in KLF2-PPAR-γ axis in modulating differentiation of macrophages will be tested. This may contribute to the exploration of new drug targets. With the preliminary studies, we are confident that KLF2 may, at a minimum, govern the macrophage polarization, modulate recruitment of inflammatory cells, prime adaptive immune response, and/or play some additional roles in disposing pathogens.
3. Approach. Conditional KLF2 knockout mouse model will be used extensively in this research. Dr. Lingrel's laboratory created and characterized this animal model in 1998 (86). LysM (murine M lyzozyme) promoter exclusively expressed in myeloid cells, drives the downstream transgene Cre expression, and then causes the recombination of loxP site that located at each end of KLF2 exon 2. We assessed the recombination and expression of KLF2 in macrophages to ensure the fidelity of gene expression. Upon the success of identifying phenotype of KLF2 knockout mice, bone marrow cells, AMs, and peritoneal exudates cells will be collected for analyzing CAMs and AAMs ratio with/without Hc challenge by flow cytometry. In addition, responsiveness and sensitivity to macrophage activation cytokines IFN-γ and IL-4 will be measured in these three different tissue specific macrophages, and macrophage phenotype, cytokine profiles, as well as receptor expressions will be assessed accordingly. In vitro studies using macrophages will, at least in part, contribute to the prediction for host response in vivo. In vivo studies are critical in determining the systemic effect in the context of KLF2 knockout mice during Hc infection. By collecting infected lung and spleen, we could measure fungal load in WT and KO, which will offer us fundamental evidence of Hc resolution under KLF2 deficient condition. Moreover, qPCR and Facs used as tools to study the cytokine profiles and cell distribution in lung will provide direct evidence of host inflammatory response in the course of infection. Together, these studies will explore the KLF2 functionality in vitro and in vivo. Further signaling pathways studies will be included in terms of correspondent findings.
