CCL7 Is a Negative Regulator of Cutaneous Inflammation Following Leishmania major Infection

The chemokine CCL7 (MCP3) is known to promote the recruitment of many innate immune cell types including monocytes and neutrophils to sites of bacterial and viral infection and eosinophils and basophils to sites of allergic inflammation. CCL7 upregulation has been associated with many inflammatory settings including infection, cardiovascular disease, and the tumor microenvironment. CCL7's pleotropic effects are due in part to its ability to bind numerous chemokine receptors, namely CCR1, CCR2, CCR3, CCR5, and CCR10. CCL7-blockade or CCL7-deficiency is often marked by decreased inflammation and poor pathogen control. In the context of Leishmania major infection, CCL7 is specifically upregulated in the skin one-2 weeks after infection but its role in L. major control is unclear. To determine CCL7's impact on the response to L. major we infected WT and CCL7−/− C57BL/6 mice. L. major infection of CCL7-deficient mice led to an unexpected increase in inflammation in the infected skin 2 weeks post-infection. A broad increase in immune cell subsets was observed but was dominated by enhanced neutrophilic infiltration. Increased neutrophil recruitment was associated with an enhanced IL-17 gene profile in the infected skin. CCL7 was shown to directly antagonize neutrophil migration in vitro and CCL7 add-back in vivo specifically reduced neutrophil influx into the infected skin revealing an unexpected role for CCL7 in limiting neutrophil recruitment during L. major infection. Enhanced neutrophilic infiltration in CCL7-deficient mice changed the balance of L. major infected host cells with an increase in the ratio of infected neutrophils over monocytes/macrophages. To determine the consequence of CCL7 deficiency on L. major control we analyzed parasite load cutaneously at the site of infection and viscerally in the draining LN and spleen. The CCL7−/− mice supported robust cutaneous parasite control similar to their WT C57BL/6 counterparts. In contrast, CCL7-deficiency led to greater parasite dissemination and poor parasite control in the spleen. Our studies reveal a novel role for CCL7 in negatively regulating cutaneous inflammation, specifically neutrophils, early during L. major infection. We propose that CCL7-mediated dampening of the early immune response in the skin may limit the ability of the parasite to disseminate without compromising cutaneous control

Inflammation-induced effector CD4+ T cell interstitial migration is alpha-v integrin dependent

Leukocytes must traverse inflamed tissues to effectively control local infection. Although motility in dense tissues appears to be integrin-independent actin-myosin based, during inflammation changes to the extracellular matrix (ECM) may necessitate distinct motility requirements. Indeed, we found that T cell interstitial motility was critically dependent on RGD-binding integrins in the inflamed dermis. Inflammation-induced deposition of fibronectin was functionally linked to increased αv integrin expression on effector CD4+ T cells. Using intravital multi-photon imaging, we found that CD4+ T cell motility was dependent on αv expression. Selective αv blockade or knockdown arrested TH1 motility in the inflamed tissue and attenuated local effector function. These data show a context-dependent specificity of lymphocyte movement in inflamed tissues that is essential for protective immunity

Modeling Susceptibility versus Resistance in Allergic Airway Disease Reveals Regulation by Tec Kinase Itk

Murine models of allergic asthma have been used to understand the mechanisms of development and pathology in this disease. In addition, knockout mice have contributed significantly to our understanding of the roles of specific molecules and cytokines in these models. However, results can vary significantly depending on the mouse strain used in the model, and in particularly in understanding the effect of specific knockouts. For example, it can be equivocal as to whether specific gene knockouts affect the susceptibility of the mice to developing the disease, or lead to resistance. Here we used a house dust mite model of allergic airway inflammation to examine the response of two strains of mice (C57BL/6 and BALB/c) which differ in their responses in allergic airway inflammation. We demonstrate an algorithm that can facilitate the understanding of the behavior of these models with regards to susceptibility (to allergic airway inflammation) (Saai) or resistance (Raai) in this model. We verify that both C57BL/6 and BALB/c develop disease, but BALB/c mice have higher Saai for development. We then use this approach to show that the absence of the Tec family kinase Itk, which regulates the production of Th2 cytokines, leads to Raai in the C57BL/6 background, but decreases Saai on the BALB/c background. We suggest that the use of such approaches could clarify the behavior of various knockout mice in modeling allergic asthma

Efficient Germinal Center Support Requires Tfh Expression of Alpha V Integrins

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2018.CD4+ T cells receive various signals and cues from their microenvironment that guide their differentiation and function as immune effectors. These signals are often crucial for shaping an immune response that is appropriate and sufficient to eliminate the causal pathogen. Many cell types rely heavily on signals provided by the extracellular matrix (ECM) for their survival, positioning, and function. Despite this, little is known about how CD4+ T cells interact with the ECM in vivo and how those interactions contribute to an immune response. The ECM in peripheral tissues as well as in secondary lymphoid organs (SLO) undergoes significant changes in structure and composition during inflammation. In addition, CD4+ T cells express a number of receptors that may be sensitive to these changes. The aim of this thesis is to evaluate how CD4+ T cells interact with the ECM and what consequences these interactions have for the immune response at large. Previous work in our lab has established that CD4+ T cells in the dermis are dependent on alpha V integrins for migration. As such, we chose to examine the role of these integrins and their RGD-containing ligands in CD4+ T cell function and positioning in the lymph node. In concert with changes in the overall structure of the ECM, RGD-containing ligands were observed to accumulate substantially during inflammation in the skin. Surprisingly, alpha V-deficient CD4+ T cells showed reduced activation capacity and cytokine production in vitro, suggesting that interaction with the ECM or a cell surface alpha V ligand may contribute to naïve cell activation and differentiation. However, compensatory mechanisms appeared to be at play in vivo allowing normal cytokine responses to inflammation. In contrast to the generalized ECM deposition observed in the skin, a spatially distinct accumulation of RGD-containing ligands was seen in the skin-draining lymph node after immunization. The alpha V ligands vitronectin, osteopontin, and MFG-E8 were detected in close proximity to the GL7+ germinal centers of the B cell follicles. Interestingly, these ECM ligands were associated with CD35+ follicular dendritic cells (FDCs) and were often concentrated in the GC light zone. Evaluation of the B cell response showed an overall reduction in GC formation resulting in lower antigen-specific antibody production, reduced affinity maturation, and poor seeding of long-lived cells to the bone marrow when CD4+ T cells lacked alpha V integrins. Surprisingly, Tfh populations appeared normal and our experiments indicated that alpha V-deficient T cells are capable of providing B cell help in vitro. Histological analysis revealed that, while alpha V-deficient T cells were present in the B cell follicles, they failed to accumulate in the alpha V ligand-rich GC light zone to the same degree as WT cells

Leishmania major shapes the early cytokine and chemokine response at the site of infection

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2007.Compartmentalization of the immune response ensures tight regulation of T cell activation in the lymph node (LN) and precise effector T cell delivery and function at sites of inflammation. During differentiation within the LN, functionally discrete effector T cells acquire distinct homing properties that direct them to sites of inflammation. We show that such tissue-specific accumulation of cytokine secreting effector T cells can be subverted by a pathogen at the infected tissue site. On Leishmania major (L. major) infection, the draining LN (dLN) of both susceptible and resistant mice contained both IL-4 and IFNγ-producing T cells early in infection. In contrast, primarily IL-4 producing cells were found in the infected tissue. Despite a striking absence of IFNγ producers in the L. major-infected tissue, L. major-specific IFNγ effectors were readily detectable at an independent inflammatory site. Recruitment of non-Leishmania specific effectors to the infection site was similarly restricted. Importantly, provision of strong inflammatory signals (CpG) failed to disrupt the selective cytokine production at the L. major infection site, suggesting that L. major actively modifies the local milieu. To formally test this idea, we assessed the ability of the L. major infected site to recruit a third-party effector population. Mice were infected with L. major in one ear and immunized with ovalbumin (OVA) in the contralateral ear. Two weeks after infection, mice were rechallenged with soluble OVA directly in the L. major infected ear. Indeed, while both anti-OVA IL-4 and IFNγ producers were found in the OVA-immunized ear and dLN, only IL-4 producing, OVA specific cells were found in the L. major-infected tissue. Mechanistically, IFNγ producers could fail to be recruited, be recruited but not be retained or be recruited and then functionally modified or terminated. Our data suggests a role for L. major in shaping the early chemokine microenvironment. Early upon infection, we observed a striking restriction in chemokine expression at the infection site; expression of CCL7 but not Th1-attracting chemokines. Moreover, L. major potently inhibited CpG-induction of various Th1-attracting chemokines in part through a direct action of the parasite on the infected macrophage. These data implicate a role for the local chemokine milieu in shaping the resulting cytokine repertoire at the site of infection. In addition we have developed techniques that will enable us to track effector cell function and localization within the whole mouse. The use of fluorescent parasites in combination with cytokine reporter mice, whole mount immunohistochemistry and bioluminescence imaging will allow us to monitor the recruitment/retention and functional status of antigen specific effector cells at the site of infection. We suggest that the pathogen-driven restriction to non-pathogenic IL-4 producing cells enables L. major to gain early host residency. This work demonstrates the need to understand the immune response at infected tissue sites in addition to the repertoire generated within the dLN. This will promote the development of therapies, which target either the generation of the appropriate immune response or direct an existing response to the appropriate location in order to combat infection

FoxP3+ Regulatory T Cell Fate in Inflamed Tissues

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2016.Regulatory CD4+ T cells (Tregs), classically defined by expression of CD25 and the transcription factor FoxP3, are immunosuppressive sentinels that function under a variety of tissues and inflammatory milieu. Tregs express different transcriptional signatures within distinct anatomical locations. However, whether the modulation of Tregs is due to distinct anatomical locations or the type of inflammation within the tissue remains unknown. We sought to define factors that govern Treg modulation in differently inflamed skin, and in healthy and autoimmune pancreas compared to lymphoid tissues. Our data reveal three major observations in Treg accumulation and function in nonlymphoid tissues in disease and under different types of inflammation. 1) Treg accumulation in non-lymphoid tissues is drastically changed depending on the type of inflammatory milieu, which may lead to changes in Treg survival or development of autoimmune diabetes. Our skin model suggests that inflammatory changes in IL-23: IL- 33 may be important in Treg accumulation and survival in tissues. In both the pancreas and skin, increased accumulation patterns correlated with a higher expression of surface CD25 on Tregs, a receptor implicated in Treg survival. Interestingly, CD25 upregulation was specific to the type of inflammatory milieu. 2) Surprisingly, gross changes in Treg transcriptional phenotype correlated more with anatomical location (skin and pancreas compared to LN) than the type of inflammation or disease state within the tissue. We identified a small number of novel transcriptional changes in Tregs associated with the diseased pancreas. 3) Inflammatory milieu directly affects Treg response to antigen in the skin. Only in inflammatory milieus that upregulated CD25 did the provision of antigen enhance local Treg proliferation. Thus, the magnitude of the Treg response in inflamed tissues is controlled at two inter-dependent levels: inflammatory signals that support the upregulation of the important Treg survival factor, CD25, and the antigen signals that drive local expansion. These results begin to reconcile some of the in vivo discrepancies about the modulation of Tregs in the non-lymphoid tissues during inflammation and autoimmune disease. Importantly, our studies may be used to advance immune therapies to govern Treg maintenance in different inflammatory and anatomical locations