Kaveh Abdi

IL-12: The Role of p40 Versus p75

Interleukin (IL)‐12p75 is a heterodimeric cytokine composed of the product of two different genes that specify p35 and p40 subunits. The prevailing view is that IL‐12 acts as a proinflammatory cytokine that bridges the innate and adaptive immune responses and skews T‐cell reactivity toward a TH1 cytokine pattern. Though the terms IL‐12 and IL‐12p40 are often used interchangeably, and measurements of the p40 chain are often interpreted as measurements of the intact p75 heterodimer, such interchangeable usage may be incorrect.

T‐cell Control of IL‐12p75 Production

It is currently thought that IL‐12, produced by dendritic cells (DC) early after stimulation by bacterial pathogens or lipopolysaccharide (LPS), acts as a pro‐inflammatory cytokine bridging the innate and adaptive immune responses. We found, however, that it is only the p40 subunit and not the IL‐12p75 heterodimer that is secreted early in copious amounts in response to LPS. Neither naïve T cells, nor a variety of microbial products, were able to induce IL‐12p75 production unless the DC were conditioned by the presence of interferon‐γ (IFN‐γ) or by encounter with previously activated T cells. The inability of naïve T cells or of bacterial products to induce IL‐12 argues against its early role as the initiator of innate and adaptive immune responses.

Lipopolysaccharide-Activated Dendritic Cells: “Exhausted” or Alert and Waiting?

LPS-activated dendritic cells (DCs) are thought to follow a set program in which they secrete inflammatory cytokines (such as IL-12) and then become refractory to further stimulation (i.e., “exhausted”). In this study, we show that mouse DCs do indeed lose their responsiveness to LPS, but nevertheless remain perfectly capable of making inflammatory cytokines in response to signals from activated T cells and to CD40-ligand and soluble T cell-derived signals. Furthermore, far from being rigidly programmed by the original activating stimulus, the DCs retained sufficient plasticity to respond differentially to interactions with Th0, Th1, Th2, and Th17 T cells. These data suggest that LPS activation does not exhaust DCs but rather primes them for subsequent signals from T cells.

Free IL-12p40 Monomer Is a Polyfunctional Adaptor for Generating Novel IL-12–like Heterodimers Extracellularly

IL-12p40 partners with the p35 and p19 polypeptides to generate the heterodimeric cytokines IL-12 and IL-23, respectively. These cytokines play critical and distinct roles in host defense. The assembly of these heterodimers is thought to take place within the cell, resulting in the secretion of fully functional cytokines. Although the p40 subunit alone can also be rapidly secreted in response to inflammatory signals, its biological significance remains unclear. In this article, we show that the secreted p40 monomer can generate de novo IL-12–like activities by combining extracellularly with p35 released from other cells. Surprisingly, an unbiased proteomic analysis reveals multiple such extracellular binding partners for p40 in the serum of mice after an endotoxin challenge. We biochemically validate the binding of one of these novel partners, the CD5 Ag-like glycoprotein, to the p40 monomer. Nevertheless, the assembled p40-CD5L heterodimer does not recapitulate the biological activity of IL-12. These findings underscore the plasticity of secreted free p40 monomer, suggesting that p40 functions as an adaptor that is able to generate multiple de novo composites in combination with other locally available polypeptide partners after secretion.

Antigen-activated T cells induce IL-12p75 production from Dendritic cells in an IFN-γ-independent manner

The addition of IL‐12p75 to naïve CD4+ T cells promotes their differentiation towards a TH1‐type cytokine pattern. Dendritic cells stimulated by LPS generate IL‐12p75, but only if the environment also contains IFN‐γ. Thus, it appears that IFN‐γ is needed to start the response that will result in further production of IFN‐γ. We previously reported that paradoxically DCs produce IL‐12p75 only after engaging primed, but not naïve T cells. This study examines the mechanism by which primed T cells trigger IL‐12p75 secretion and asks whether this induction is also dependent on the presence of IFN‐γ. Here, we show that, in contrast to LPS, primed T cells induce IL‐12p75 in an IFN‐γ‐independent manner. Addition of rIFN‐γ to cocultures of naïve T cells with DCs did not induce IL‐12p75. Moreover, antigen‐activated CD4+ T cells from wild type or IFN‐γ‐deficient mice both initiated IL‐12p75 production from DCs. Surprisingly, we found that synergies between three T‐cell‐derived factors – CD40 Ligand, IL‐4 and GM‐CSF – were necessary and sufficient for IL‐12p75 production. These results suggest that there are at least two distinct pathways for IL‐12p75 production in vivo. Furthermore, the T‐cell‐dependent pathway of IL‐12p75 production employs molecules that are not classically associated with a TH1‐type response.

Mechanisms by which Porphyromonas gingivalis evades innate immunity

The oral cavity is home to unique resident microbial communities whose interactions with host immunity are less frequently studied than those of the intestinal microbiome. We examined the stimulatory capacity and the interactions of two oral bacteria, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum), on Dendritic Cell (DC) activation, comparing them to the effects of the well-studied intestinal microbe Escherichia coli (E. coli). Unlike F. nucleatum and E. coli, P. gingivalis failed to activate DCs, and in fact silenced DC responses induced by F. nucleatum or E. coli. We identified a variant strain of P. gingivalis (W50) that lacked this immunomodulatory activity. Using biochemical approaches and whole genome sequencing to compare the two substrains, we found a point mutation in the hagA gene. This protein is though to be involved in the alteration of the PorSS/gingipain pathway, which regulates protein secretion into the extracellular environment. A proteomic comparison of the secreted products of the two substrains revealed enzymatic differences corresponding to this phenotype. We found that P. gingivalis secretes gingipain(s) that inactivate several key proinflammatory mediators made by DCs and/or T cells, but spare Interleukin-1 (IL-1) and GM-CSF, which can cause capillary leaks that serve as a source of the heme that P. gingivalis requires for its survival, and GM-CSF, which can cause epithelial-cell growth. Taken together, our results suggest that P. gingivalis has evolved potent mechanisms to modulate its virulence factors and dampen the innate immune response by selectively inactivating most proinflammatory cytokines.

Cutting Edge: Quantitative Determination of CD40L Threshold for IL-12 and IL-23 Production from Dendritic Cells

Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-γ. However, only activated, but not naive T cells are able to license DCs for IL-12 production. We hypothesized that it might be due to different levels of CD40L expression on the surface of these cells, as CD40 signals are required for IL-12 production. Using quantitative cell-free systems incorporating CD40L in lipid bilayers combined with total internal reflection fluorescence microscopy and flow cytometry, we show that as low as ∼200 CD40L molecules/μm2 in combination with IL-4 is sufficient to induce IL-12 production by DCs. Remarkably, CD40L alone is adequate to induce IL-23 secretion by DCs. Thus, although activated T cells have somewhat higher levels of CD40L, it is the combination of CD40L and the cytokines they secrete that licenses DCs and influences the effector class of the immune response.